Malaria vaccine

ABSTRACT

The present invention features immunogenic compositions based on pre-fertilization or post-fertilization antigens expressed in the circulating gametocytes in the peripheral blood of infected persons or on the malaria parastes&#39; stages of develop-ment in the mosquito midgut including extracellular male and female gametes, fertilized zygote and ookinete. The invention also features methods to prevent the transmission of malaria using the immunogenic compositions of the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/099,651, which was filed on Sep. 24, 2008, the entire contents ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Malaria is one of most dangerous infectious diseases in tropical andsubtropical countries, afflicting about 300 million people. The pathogenof the disease is a protozoan parasite, Plasmodium sp. which istransmitted by Anopheles mosquitoes. Four species of malaria parasitescan infect humans under natural conditions: Plasmodium falciparum, P.vivax, P. ovale, and P. malariae. P. falciparum and P. vivax cause themost infections worldwide.

P. falciparum is the agent of severe, potentially fatal malaria. Malariacaused by P. falciparum is responsible for nearly 1 million deathsannually. Based on recent estimates from the WHO, worldwide, there werean estimated 247 million malaria cases among 3.3 billion people at riskliving in 109 countries [1]. Infections caused by P. falciparum and P.vivax account for more than 90% of global malaria burden; the formerbeing responsible for nearly all the deaths due to malaria, nearly amillion deaths of children under 5 years [2]. Among the current effortsagainst malaria include increasing use of insecticide treated bed netsand use of combination drugs to tackle the problem associated with drugresistance. The emergence of drug-resistant strains over the last 4decades has underscored the necessity for improved control strategies.Accordingly, the development of a safe and effective malaria vaccinewill be an important step towards controlling malaria. Vaccinedevelopment efforts to date have focused on candidate antigensrepresented in the pre-erythrocytic, erythrocytic and sexual stages ofthe parasite. Currently, the only vaccine advanced in clinicaldevelopment, RTS,S, has shown partial protection against infection anddisease severity in several clinical trials [6,7].

Immunity against the sexual stages of the parasite offers an effectiveway to reduce or stop malaria transmission and in that respect offersthe greatest promise towards the goal of progressively eliminatingmalaria from endemic countries. A transmission blocking vaccine (TBV)[8] specifically targeting the sexual development of the parasite in themosquito vector may elicit immunity which can effectively blocktransmission of the parasite from invertebrate mosquito vector tovertebrate host. Transmission of malaria depends upon the presence ofinfectious male and female gametocytes in the peripheral blood ofinfected persons and successful ingestion of these gametocytes byAnopheles mosquitoes. Soon after ingestion, exflagellation occurs withinthe mosquito midgut, and emergent male gametes fertilize female gametes,resulting in the formation of zygotes. The zygotes undergopost-fertilization transformation into motile ookinetes which traversethe midgut epithelium and develop into oocysts resulting in theproduction of infective sporozoites. Finally the sporozoites arereleased into the hemocoel, invade the salivary glands and aretransmitted to vertebrate hosts during subsequent blood feeding [9].

The targets of transmission blocking antibodies includepre-fertilization antigens (Pfs230 and Pfs48/45) expressed in thecirculating gametocytes and post-fertilization antigens (Pfs25 andPfs28) expressed during mosquito stage ookinete development [10]. UnlikePfs25 and Pfs28, pre-fertilization antigens are also targets of thenatural immune response and thus immunity induced by a vaccine based onany of these antigens will have the added benefit of natural boosting ofimmunity. To date, only Pfs25 and Pvs25 (P. vivax homolog of Pfs25) haveundergone limited Phase I clinical trials with marginal success [11,12].It has not been possible to evaluate any of the pre-fertilizationantigens as vaccines simply because they have not been available insufficient quantity and proper protein conformation.

Although much progress has been made in the recent past, the developmentof a safe, effective and affordable malaria vaccine has remained achallenge. A vaccine targeting sexual stages of the parasite will notonly reduce malaria transmission by female Anopheles mosquitoes, butalso reduce the spread of parasites able to evade immunity elicited byvaccines targeting pre-erythrocytic and erythrocytic asexual stages.

SUMMARY OF THE INVENTION

As described below, the present invention features immunogeniccompositions based on pre-fertilization antigens expressed in thecirculating gametocytes in the peripheral blood of infected persons. Thepresent invention makes use of an approach that harmonizes codon usagefrequency of the target gene with those of the expression host forheterologous expression of protein. Taking these concepts into account,an algorithm termed “codon harmonization” [19] was developed wheresynonymous codons from E. coli were selected that closely resemble thecodon usage of a native pre-fertilization gene, for example the nativePfs48/45 gene, including regions coding ‘link/end’ segments of proteinsin P. falciparum.

Also featured in the invention are antibodies, and methods to preventthe transmission of malaria using the immunogenic compositions of theinvention.

In a first aspect, the invention features a method of blockingtransmission of Plasmodium falciparum or Plasmodium vivax in a subjectcomprising administering to a subject an immunogenic compositioncomprising one or more Plasmodium falciparum or Plasmodium vivaxpre-fertilization antigens, thereby blocking transmission of Plasmodiumfalciparum or Plasmodium vivax in the subject.

In another aspect, the invention features a method of immunizing asubject against Plasmodium falciparum or Plasmodium vivax comprisingadministering to a subject an immunogenic composition comprising one ormore Plasmodium falciparum or Plasmodium vivax pre-fertilizationantigens, thereby immunizing the subject against Plasmodium falciparumor Plasmodium vivax.

In still another aspect, the invention features a method for treating orpreventing malaria in a subject comprising administering to a subject animmunogenic composition comprising one or more Plasmodium falciparum orPlasmodium vivax pre-fertilization antigens, thereby preventing malariain the subject.

In another aspect, the invention features a method of blockingtransmission of Plasmodium falciparum or Plasmodium vivax in a subjectcomprising administering to a subject an immunogenic compositioncomprising one or more Plasmodium falciparum or Plasmodium vivaxpost-fertilization antigens, thereby blocking transmission of Plasmodiumfalciparum or Plasmodium vivax in the subject.

In one aspect, the invention features a method of immunizing a subjectagainst Plasmodium falciparum or Plasmodium vivax comprisingadministering to a subject an immunogenic composition comprising one ormore Plasmodium falciparum or Plasmodium vivax post-fertilizationantigens, thereby immunizing the subject against Plasmodium falciparumor Plasmodium vivax.

In another aspect, the invention features a method for treating orpreventing malaria in a subject comprising administering to a subject animmunogenic composition comprising one or more Plasmodium falciparum orPlasmodium vivax post-fertilization antigens, thereby preventing malariain the subject.

In one embodiment of any one of the above aspects, the one or morepre-fertilization or post-fertilization antigens are selected from thegroup consisting of Pfs48/45, Pfs 230, Pfs25, Pvs48/45, Pvs 230 andPvs25.

In a further embodiments, the pre-fertilization antigen is Pfs48/45.

In another further embodiment, the post-fertilization antigen is Pfs25.

In another embodiment of any one of the above aspects, the one or morepre-fertilization antigens or post-fertilization antigens is derivedfrom a codon harmonized gene. In a particular embodiment, the codonharmonized gene is encoded by the amino acid sequence corresponding toSEQ ID NO: 1. In another particular embodiment, the codon harmonizedgene is encoded by the amino acid sequence corresponding to SEQ ID NO:3. In still another particular embodiment, the codon harmonized gene isencoded by the amino acid sequence corresponding to SEQ ID NO: 5.

In another aspect, the present invention features a method of blockingtransmission of Plasmodium falciparum or Plasmodium vivax in a subjectcomprising administering to a subject an immunogenic compositioncomprising one or more Plasmodium falciparum or Plasmodium vivax surfaceantigens, thereby blocking transmission of Plasmodium falciparum orPlasmodium vivax in the subject.

In another preferred aspect, the invention features a method ofimmunizing a subject against Plasmodium falciparum or Plasmodium vivaxcomprising administering to a subject an immunogenic compositioncomprising one or more Plasmodium falciparum or Plasmodium vivax surfaceantigens, thereby immunizing the subject against Plasmodium falciparumor Plasmodium vivax.

In still another aspect, the invention features a method for treating orpreventing malaria in a subject comprising administering to a subject animmunogenic composition comprising one or more Plasmodium falciparum orPlasmodium vivax surface antigens, thereby preventing malaria in thesubject.

In one embodiment of any one of the above aspects, the surface antigensare gametocyte or gamete surface antigens.

In a related embodiment, the gametocyte or gamete surface antigens areselected from the group consisting of: Pfs48/45, Pfs230, Pvs48/45 andPvs230.

In another further embodiment of any one of the above aspects, thesurface antigens are midgut parasite surface antigens. In a relatedembodiment, the midgut parasite surface antigens are selected from thegroup consisting of: Pfs25, Pfs28, Pvs25 and Pvs28.

In still another further embodiment of any one of the above aspects, theone or more surface antigens is derived from a codon harmonized gene.

In another related embodiment, the codon harmonized gene is encoded bythe amino acid sequence corresponding to SEQ ID NO: 1. In anotherembodiment, the codon harmonized gene is encoded by the amino acidsequence corresponding to SEQ ID NO: 3. In another further embodiments,the codon harmonized gene is encoded by the amino acid sequencecorresponding to SEQ ID NO: 5.

In another further embodiment of any one of the above aspects, blockingtransmission is measured by the reduction of mosquito oocysts by sera orplasma from a subject treated with the immunogenic composition comparedto a control subject. In a further related embodiment, pre-immune serafrom the treated subject and the control subject are used as a measureof 100% transmission for the corresponding test sera.

In another further embodiment of any one of the above aspects, themethod further comprises administering an adjuvant. In a relatedembodiment, the adjuvant is selected from water-in-oil emulsion orAluminum hydroxide.

In another further embodiment of any one of the above aspects, themethod further comprises administering the immunogenic composition inone or more booster administrations. In a particular embodiment, thebooster immunization is administered at 4 weeks. In another particularembodiment, the booster immunization is administered at 12 weeks.

In another further embodiment of any one of the above aspects, themethod further comprises treatment with an additional agent. In afurther embodiment, the additional agent is used to treat or preventmalaria.

In another further embodiment of any one of the above aspects, thecomposition is administered by one or more routes selected from thegroup consisting of: subcutaneous, intradermal, intramuscular,intravenous and transdermal delivery.

In still another further embodiment of any one of the above aspects, thecomposition is administered in a concentration between 1-100 μg.

In another aspect, the invention features an immunogenic compositioncomprising one or more pre-fertilization or post-fertilization antigensfrom P. falciparium or P. vivax.

In one embodiment, the one or more pre-fertilization orpost-fertilization antigens are selected from the group consisting ofPfs48/45, Pfs230, Pfs 28, Pfs25, Pvs48/45, Pvs 230, Pvs 28, and Pvs25.

In a particular embodiment, the pre-fertilization antigen is Pfs48/45

In another particular embodiment, the post-fertilization antigen isPfs25.

In still another further embodiment, the one or more pre-fertilizationor post-fertilization antigens is derived from a codon harmonized gene.

In a further related embodiment, the codon harmonized gene encodes aprotein represented by the amino acid sequence corresponding to SEQ IDNO: 1. In another embodiment, the codon harmonized gene is encoded bythe amino acid sequence corresponding to SEQ ID NO: 3. In anotherfurther embodiment, the codon harmonized gene is encoded by the aminoacid sequence corresponding to SEQ ID NO: 5.

In another further embodiment of any one of the above aspects, thecomposition is designed for expression in E. coli.

In another aspect, the invention features a vector comprising a codonharmonized Pfs48/45 sequence suitable for expression in a cell.

In still another aspect, the invention features a vector comprising acodon harmonized Pvs48/45 sequence suitable for expression in a cell.

In yet another aspect, the invention features a vector comprising acodon harmonized Pfs25 sequence suitable for expression in a cell.

In one embodiment, the codon harmonized Pfs48/45 sequence corresponds tothe nucleic acid sequence comprising SEQ ID NO: 2. In anotherembodiment, the codon harmonized Pfs25 sequence corresponds to thenucleic acid sequence comprising SEQ ID NO: 4. In still anotherembodiment, the codon harmonized Pvs48/45 sequence corresponds to thenucleic acid sequence comprising SEQ ID NO: 6.

In one embodiment of any one of the above aspects, the inventionfeatures a cell expressing the vector of any one of the aspectsdescribed.

In one embodiment, the cell is an E. coli cell or an E. coli derivativecell.

In another aspect, the invention features a kit comprising animmunogenic composition comprising one or more Plasmodium falciparum orPlasmodium vivax pre-fertilization or post-fertilization antigens, andinstructions for use in reducing transmission of Plasmodium falciparumor Plasmodium vivax.

In yet another aspect, the invention features a kit comprising animmunogenic composition comprising one or more Plasmodium falciparum orPlasmodium vivax pre-fertilization or post-fertilization antigens, andinstructions for use in preventing malaria.

In one embodiment, the one or more pre-fertilization orpost-fertilization antigens are selected from the group consisting ofPfs48/45, Pfs230, Pfs 28, Pfs25, Pvs48/45, Pvs 230, Pvs 28, and Pvs25.

In another particular embodiment, the one or more pre-fertilization orpost-fertilization antigens is derived from a codon harmonized gene.

In another aspect, the invention features a method for preparing a codonharmonized pre-fertilization or post-fertilization antigen sequenceencoded by a P. falciparum or P. vivax pre-fertilization orpost-fertilization gene comprising determining the frequency of codonusage of the pre-fertilization or post-fertilization gene codingsequence; and substituting codons in the coding sequence with codons ofsimilar frequency from a host cell which code for the samepre-fertilization or post-fertilization antigen, thereby preparing acodon harmonized pre-fertilization or post-fertilization antigensequence.

In one embodiment, the one or more pre-fertilization orpost-fertilization genes are selected from the group consisting ofPfs48/45, Pfs230, Pfs 28, Pfs25, Pvs48/45, Pvs 230, Pvs 28, and Pvs25.

In another aspect, the invention features a method for preparing a codonharmonized Pfs48/45 antigen sequence encoded by a pre-fertilization orpost-fertilization gene comprising determining the frequency of codonusage of the pre-fertilization or post-fertilization gene codingsequence, wherein the Pfs48/45 sequence corresponding to the nucleicacid sequence represented by SEQ ID NO: 7, and substituting codons inthe coding sequence of SEQ ID NO: 7 with codons of similar frequencyfrom a host cell which code for the Pfs48/45 antigen, thereby preparinga codon harmonized Pfs48/45 antigen sequence.

In one embodiment, the codon harmonized gene is expressed in a hostcell.

In another embodiment, the host cell is E. coli or an E. coliderivative.

In still another embodiment, the invention features a codon harmonizednucleotide sequence prepared according to the methods described herein.

Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying drawings,incorporated herein by reference.

Various preferred features and embodiments of the present invention willnow be described by way of non-limiting example and with reference tothe accompanying drawings in which:

FIGS. 1 (a-f) shows the purification and characterization ofCH-rPfs48/45. (a) Schematic representation of amino acid residues in therecombinant protein expressed. First 16 amino residues at the N-terminuscontain 6× Histidine residues and a short linker to allow affinitypurification of the protein. The sequence of Pfs48/45 begins withasparagine (N) at the 28 position and ends with serine (S) at 427position in the native Pfs48/45 sequence. (b) Induction profile ofCH-rPfs48/45. The cells were induced with 0.1 mM IPTG for 3 h and lysedby sonication. Lysates of uninduced and induced cells either non-reduced(left panel), or reduced (treated with 10 mM 2-mercaptoethanol, rightpanel) were run on SDS-polyacrylamide gel and stained with Coomassiestain. Lane 1; Protein molecular weight marker; lane 2, uninduced celllysate; lane 3, induced cell lysate. The induced protein band in bothnon-reduced and reduced gels are encircled for easy recognition. (c)Flow diagram of various major steps including differential detergentextractions used for protein purification. (d) Western blot analysis forthe presence of CH-rPfs48/45 at each step of detergent extraction usinganti-His mAb. Lane 1, lysate pellet; lane 2, lysate supernatant; lane 3,1% Tween-80 pellet; lane 4, 1% Tween-80 supernatant; lane 5, 0.5%sarcosyl pellet; lane 6, 0.5% sarcosyl supernatant. (e) Western blotanalysis of purified CH-rPfs48/45 using anti-His mAb. Lane 1, eluatefrom Ni-NTA column; lane 2, dialyzed CH-rPfs48/45. (f) Recognition ofCH-rPfs48/45 by conformation specific mAb IIC5B10. Lane 1, non-reducedCH-rPfs48/45; lane 2, reduced CH-rPfs48/45.

FIGS. 2 (a & b) is two panels that show (a) ELISA analysis ofCH-rPfs48/45 immunized individual mouse sera in three different adjuvantformulations: Complete Freund's adjuvant (top panel), Montanide ISA-51(middle panel), and Alum (bottom panel). All the results arerepresentative of three independent experiments. Pooled pre-immune sera+3SD are shown by broken lines. ELISA OD405 values for individual miceare shown; mouse 1 (filled triangle), mouse 2 (open square), mouse 3(filled square), mouse 4 (open circle), mouse 5 (filled circle). (b)Analysis of anti-Pfs48/45 IgG isotype distribution in individual mousesera: IgG1 (filled columns), IgG2a (hatched columns), IgG2b (stippledcolumns), IgG3 (blank columns).

FIGS. 3 (a & b) shows recognition of native Pfs48/45 in P. falciparumgametocyte extract. (a) Western blot analysis with non-reduced (leftpanel) or reduced (right panel) P. falciparum gametocyte extract againstserum of individual mouse immunized with either CFA or ISA-51 or alumformulation. Stage V gametocyte extract was run either in non-reduced orreduced (10 mM 2-mercaptothanol) form in SDS-PAGE and transferred tonitrocellulose membrane. Mice sera were allowed to react at 1:1000dilution for 1 h at 22° C. HRP-conjugated anti-mouse IgG at 1:10000dilution was used as detection antibody and was developed using ECLsubstrate. Lane 1, mAb IIC5B10; lane 2, one representative mouse serumimmunized in CFA; lane 3, one representative mouse serum immunized inMontanide ISA-51; lane 4, one representative mouse serum immunized inalum formulation. The figure is assembled from separate experiments. (b)Mouse sera (1:1000 dilution) were tested by live immunofluorescenceassays as described under materials and methods.

FIGS. 4 (a-c) shows analysis of anti-Pfs48/45 antibody production byOlive baboons (Papio anubis). (a) Each animal was immunized withCH-rPfs48/45 (50 μg in 0.25 ml endotoxin free PBS) formulated inMontanide ISA-51 (0.25 ml) in baboons, administered intra muscularly(quadriceps, two sites). Schedules for immunization and bleeds areindicated and sera were stored at −20° C. until shipped frozen fromKenya to Baltimore for ELISA and MFA. The samples were shipped under anexport permit CITES # 008101. (b) Anti-Pfs48/45 whole IgG titer atvarious time points analyzed by ELISA. Pre-immune +3×SD is shown bysolid horizontal lines. ELISA readings with sera dilutions, 1 month postprimary immunization (filled square), 1 month post first boost (filledtriangle) and 1 month post second boost (filled diamond) are shown with±SD for individual baboons (Pan 3104, Pan 3140, Pan 3163, Pan 3275, Pan3313). (c) Distribution of anti-Pfs48/45 IgG1(solid diamonds) and IgG2(open diamonds) subtypes in 1 month post primary immunization sera (Dec10), 1 month post 1st boost (Jan 10), 1 month post 2nd boost (Mar 06),and 3 months post 2nd boost (May 5). Data are presented as mean OD405value±SD for individual baboon.

FIG. 5 is a graph that shows follow up of immune responses elicited byCH-rPfs48/45 in baboons. Analysis of anti-CH-rPfs48/45 IgG titers (openbars) and percent transmission blocking activity (closed bars) up to 7months post second boost in baboons. Results show mean antibody titerand mean transmission blocking activity of 5 baboons+95% CI.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, for example,hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine,phosphothreonine.

The term “codon harmonization” is meant to refer to a process thatharmonizes codon usage frequency of a target gene with those of theexpression host for heterologous expression of protein. In preferredembodiments, codon harmonization refers to an algorithm where synonymouscodons from E. coli are selected that closely resemble the codon usageof a native pre-implantation gene, for example the Pfs48/45 gene,including regions coding ‘link/end’ segments of proteins in P.falciparum.

By “fragment” is meant a portion (e.g., at least 10, 25, 50, 100, 125,150, 200, 250, 300, 350, 400, or 500 amino acids or nucleic acids) of aprotein or nucleic acid molecule that is substantially identical to areference protein or nucleic acid and retains the biological activity ofthe reference. In some embodiments the portion retains at least 50%,75%, or 80%, or more preferably 90%, 95%, or even 99% of the biologicalactivity of the reference protein or nucleic acid described herein.

The term “host cell” is meant to refer to a cell into which a foreigngene is introduced. The host cell can be prokaryotic or eukaryotic. Inpreferred embodiments, the host cell is E. coli or an E. coliderivative.

By “immunogenic composition” is meant to refer to one or more Plasmodiumpre-fertilization or post-fertilization antigens that is capable ofeliciting protection against malaria, whether partial or complete. Animmunogenic composition may also be useful for treatment of an infectedindividual.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. Various levels of purity maybe applied as needed according to this invention in the differentmethodologies set forth herein; the customary purity standards known inthe art may be used if no standard is otherwise specified.

By “isolated nucleic acid molecule” is meant a nucleic acid (e.g., aDNA, RNA, or analog thereof) that is free of the genes which, in thenaturally-occurring genome of the organism from which the nucleic acidmolecule of the invention is derived, flank the gene. The term thereforeincludes, for example, a recombinant DNA that is incorporated into avector; into an autonomously replicating plasmid or virus; or into thegenomic DNA of a prokaryote or eukaryote; or that exists as a separatemolecule (for example, a cDNA or a genomic or cDNA fragment produced byPCR or restriction endonuclease digestion) independent of othersequences. In addition, the term includes an RNA molecule which istranscribed from a DNA molecule, as well as a recombinant DNA which ispart of a hybrid gene encoding additional polypeptide sequence.

By “nucleic acid” is meant an oligomer or polymer of ribonucleic acid ordeoxyribonucleic acid, or analog thereof. This term includes oligomersconsisting of naturally occurring bases, sugars, and intersugar(backbone) linkages as well as oligomers having non-naturally occurringportions which function similarly. Such modified or substitutedoligonucleotides are often preferred over native forms because ofproperties such as, for example, enhanced stability in the presence ofnucleases.

The term “complimentary nucleic acid sequences” refer to contiguous DNAor RNA sequences which have compatible nucleotides (e.g., A/T, G/C) incorresponding positions, such that base pairing between the sequencesoccurs. For example, the sense and anti-sense strands of adouble-stranded DNA helix are known in the art to be complimentary.

By “pre- or post-fertilization antigen” is meant to refer to a proteintarget expressed in Plasmodium that is necessary for Plasmodiumtransmission in a host. In particular embodiments, prefertilizationantigens refer to antigens expressed in the intraerythrocytic gamtocytesand male and female gametes prior to fertilization process. Examplesinclude, but are not limited to, proteins such as Pfs230 and Pfs48/45 inP. falciparum or Pvs230 and Pvs48/45 in P. vivax. In other particularembodiments, post fertilization antigens refer to antigens expressed onthe surface of zygotes formed after fertilization between female andmale gametes and ookinetes. Examples include, but are not limited to,proteins such as Pfs25 and Pfs28 in P. falciparum and Pvs25 and Pvs28 inP. vivax.

By “protein” is meant any chain of amino acids, or analogs thereof,regardless of length or post-translational modification.

By “reference” is meant a standard or control condition.

By “specifically binds” is meant a molecule (e.g., peptide,polynucleotide) that recognizes and binds a protein or nucleic acidmolecule of the invention, but which does not substantially recognizeand bind other molecules in a sample, for example, a biological sample,which naturally includes a protein of the invention.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith, for example malaria. It will be appreciated that, althoughnot precluded, treating a disorder or condition does not require thatthe disorder, condition or symptoms associated therewith be completelyeliminated.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition,for example malaria.

Other definitions appear in context throughout the disclosure.

Compositions

The present invention features immunogenic compositions comprising oneor more pre-fertilization or post-fertilization antigens fromPlasmodium, preferably P. falciparum or P. vivax. The genome ofPlasmodium falciparum has been completely sequenced. (Gardner et al.,Nature, 419:498-511 (2002)).

Preferably, the one or more of pre-fertilization or post-fertilizationantigens are selected from the group consisting of Pfs48/45, Pfs230, Pfs28, Pfs25, Pvs48/45, Pvs 230 Pvs 28, and Pvs25. Most preferably, theantigen is Pfs 48/45 or Pfs25 of P. falciparum. Alternatively, theantigen is Pvs 48/45 or Pvs25 of P. vivax.

Pfs230 and Pfs48/45 can be classified as gametocyte and gamete (male andfemale) surface antigens. Pfs25 and Pfs28 can be classified as mosquitomidgut parasite stages (zygote and ookinete) surface antigens.

Targeted gene disruption studies have shown that Pfs48/45 plays acritical role in male gamete fertility, an important aspect of thesexual reproduction success of the parasite [14]. Analysis of immunehuman sera in endemic areas has also suggested a strong correlationbetween naturally present anti-Pfs48/45 antibodies and transmissionreducing activity of those human sera; thus making it a key candidatefor vaccine development [15]. However, efforts to produce full lengthrecombinant Pfs48/45 in a functional conformation have largely remainedunsuccessful. In a recent study, an approach that involved co-expressionof a truncated version of C-terminal fragment of Pfs48/45 fused with alarge fusion partner maltose binding protein (MBP) along with fourperiplasmic folding catalysts DsbA, DsbC, FkpA and SurA resulted incorrectly folded truncated product which produced high titers oftransmission-reducing antibodies in immunized BALB/c mice [16]. In thisrecombinant expression approach periplasmic targeting and folding intofunctional conformation of expressed Pfs48/45 protein was strictlydependent upon fusion with MBP as a carrier protein and protein foldingwas catalyzed by four chaperons co-expressed in the host, respectively.

Pfs25 is a P. falciparum antigen expressed on the surface of the malariaparasite. Pfs25 consists of four epidermal growth factor (EGF)-likedomains located between a secretory signal sequence at the N-terminus,and a short C-terminal hydrophobic domain, which seems involved in thetransfer of the EGF-like domains to a glycosyl-phosphatidylinositol(GPI) lipid anchor. There are 22 cysteine residues present as disulfidebonds in the four EGF-like domains of Pfs25. (Kaslow, et al., Trends inBiotechnology, 10:388-391 (1992); Kaslow, et al., Nature, 333:74-76(1988)). The disulfide bonds between the cysteine residues are essentialfor maintaining the structural integrity of the antigen. In an ex vivoexperiment, antibodies to the antigen can completely block transmissionof P. falciparum. (Vermeulen, et al., J. Exp. Med. 162:1460-1476 (1985))The counterpart of Pfs25 in P. vivax is Pvs25.

It is a novel feature of the present invention that the one or morepre-fertilization or post-fertilization antigens is derived from a codonharmonized gene.

Codon Harmonization

It has been discovered that a nucleotide sequence capable of enhancedexpression in host cells can be obtained by harmonizing the frequency ofcodon usage in the foreign gene at each codon in the coding sequence tothat used by the host cell.

In certain embodiments, the invention features a nucleic acid sequenceencoding a polypeptide to enhance expression and accumulation of thepolypeptide in the host cell. Accordingly, the present inventionprovides novel nucleic acid sequences, encoding a polypeptide or proteinthat is foreign to a host cell, and that is expressed at greater levelsand with greater biological activity than in the host cell as comparedto the wild-type sequence if expressed in the same host cell.

Certain examples of codon harmonization have been described, forexample, in US Application Nos. 20060088547 and 20080076161 and Angov etal. (PLOS, 2008. Volume 3, issue 5), which are incorporated by referencein their entireties herein.

The methods of the present invention, while directed to codonharmonization of pre- or post-fertilization antigens, are not limited assuch, and are applicable to any coding sequence encoding a proteinforeign to a host cell in which the protein is expressed.

Accordingly, in certain embodiments the invention features a method forpreparing a codon harmonized pre-fertilization or post-fertilizationantigen sequence encoded by P. falciparum or P. vivax apre-fertilization or post-fertilization gene comprising determining thefrequency of codon usage of the pre-fertilization or post-fertilizationgene coding sequence, and substituting codons in the coding sequencewith codons of similar frequency from a host cell which code for thesame pre-fertilization or post-fertilization antigen, thereby preparinga codon harmonized pre-fertilization or post-fertilization antigensequence.

For example, the frequency of occurrence of each codon in the P.falciparum or P. vivax pre-fertilization or post-fertilization gene ofinterest can be calculated and replaced with an E. coli codon with asimilar frequency for the same amino acid. Preferably, the one or morepre-fertilization or post-fertilization genes are selected from thegroup consisting of Pfs48/45, Pfs230, Pfs 28, Pfs25, Pvs48/45, Pvs 230,Pvs 28, and Pvs25.

An existing DNA sequence can be used as the starting material andmodified by standard mutagenesis methods that are known to those skilledin the art or a synthetic DNA sequence having the desired codons can beproduced by known oligonucleotide synthesis, PCR amplification, and DNAligation methods.

For example, the invention features a method for preparing a codonharmonized Pfs48/45 antigen sequence encoded by a pre-fertilization orpost-fertilization gene comprising determining the frequency of codonusage of the pre-fertilization or post-fertilization gene codingsequence, wherein the Pfs48/45 sequence corresponding to the nucleicacid sequence represented by SEQ ID NO: 7, and substituting codons inthe coding sequence of SEQ ID NO: 7 with codons of similar frequencyfrom a host cell which code for the Pfs48/45 antigen, thereby preparinga codon harmonized Pfs48/45 antigen sequence.

The native sequence of Pfs48/45 is represented by NCBI accession numberAF356146, corresponding to SEQ ID NO: 7 shown below:

SEQ ID NO: 7   1 atgatgttat atatttctgc gaaaaaggct caagttgctt tcatcttata tatagtattg  61 gtattaagaa taataagtgg aaacaatgat ttttataatc ctagcgcttt gaatagtgaa 121 atatctggat ttataggata taagtgtaat ttttcaaatg aaggtgttca taatttaaag 181 ccagatatgc gtgaacgtag gtctattttt tgcaacatcc attcgtattt tatatatgat 241 aagataagat taataatacc taaaaaaagt tcgtcacctg agtttaaaat attacccgaa 301 aaatgttttc aaaaagtata tactgattat gagaatagag ttgaaactga tatatcggaa 361 ttaggtttaa ttgaatatga aatagaagaa aatgatacaa accctaatta taatgaaagg 421 acaataacta tatctccatt tagtccaaaa gacattgaat ttttttgttt ttgtgataat 481 actgaaaagg ttatatcaag tatagaaggg agaagtgcta tggtacatgt acgtgtatta 541 aaatatccac ataatatttt atttactaat ttaacaaatg atctttttac atatttgccg 601 aaaacatata atgaatctaa ttttgtaagt aatgtattag aagtagaatt aaatgatgga 661 gaattatttg ttttagcttg tgaactaatt aataaaaaat gttttcaaga aggaaaagaa 721 aaagccttat ataaaagtaa taaaataatt tatcataata agttaactat ctttaaagct 781 ccattttatg ttacatcaaa agatgttaat acagaatgta catgcaaatt taaaaataat 841 aattataaaa tagttttaaa accaaaatat gaaaaaaaag tcatacacgg atgtaacttc 901 tcttcaaatg ttagttctaa acatactttt acagatagtt tagatatttc tttagttgat 961 gatagtgcac atatttcatg taacgtacat ttgtctgaac caaaatataa tcatttggta1021 ggtttaaatt gtcctggtga tattatacca gattgctttt ttcaagtata tcaacctgaa1081 tcagaagaac ttgaaccatc caacattgtt tatttagatt cacaaataaa tataggagat1141 attgaatatt atgaagatgc tgaaggagat gataaaatta aattatttgg tatagttgga1201 agtataccaa aaacgacatc ttttacttgt atatgtaaga aggataaaaa aagtgcttat1261 atgacagtta ctatagattc agcatattat ggatttttgg ctaaaacatt tatattccta1321 attgtagcaa tattattata tatttag

DNA sequences modified by the method of the present invention areexpressed at a greater level in host cells than the correspondingnon-modified DNA sequence. Preferably, the host cell is E. coli or an E.coli derivative. The method can be applied to any DNA sequence forintroduction into a host cell to provide protein product.

In particular exemplary embodiments of the present invention, the codonharmonized gene corresponds to the amino acid sequence corresponding toSEQ ID NO: 1, shown below, and the corresponding nucleic acid sequence,SEQ ID NO: 2.

SEQ ID NO: 1 NNDFYNPSALNSEISGFIGYKCNFSNEGVHNLKPDMRERRSIFCNIHSYFIYDKIRLIIPKKSSSPEFKILPEKCFQKVYTDYENRVETDISELGLIEYEIEENDTNPNYNERTITISPFSPKDIEFFCFCDNTEKVISSIEGRSAMVHVRVLKYPHNILFTNLTNDLFTYLPKTYNESNFVSNVLEVELNDGELFVLACELINKKCFQEGKEKALYKSNKIIYHNKLTIFKAPFYVTSKDVNTECTCKFKNNNYKIVLKPKYEKKVIHGCNFSSNVSSKHTFTDSLDISLVDDSAHISCNVHLSEPKYNHLVGLNCPGDIIPDCFFQVYQPESEELEPSNIVYLDSQINIGDIEYYEDAEGDDKIKLFGIVGSIP KTTSFTCICKKDKKSAYMTVTIDSSEQ ID NO: 2 AATAACGACTTCTACAACCCATCGGCTCTCAACTCTGAAATCAGCGGCTTCATCGGCTACAAGTGCAACTTCAGCAACGAAGGCGTTCACAACCTGAAGCCAGACATGCGAGAACGACGCAGCATTTTCTGTAATATACACTCGTACTTCATCTACGACAAGATCCGTCTGATCATCCCAAAAAAAAGCTCGAGCCCAGAGTTCAAAATCCTGCCTGAAAAATGCTTCCAGAAAGTTTACACTGACTACGAGAACCGTGTTGAAACTGACATCTCGGAACTGGGCCTGATTGAATACGAAATCGAAGAAAACGACACCAATCCAAACTACAACGAACGCACCATCACGATCAGCCCATTCTCTCCAAAAGATATTGAATTCTTCTGCTTCTGCGACAACACTGAAAAGGTTATCAGCTCTATCGAAGGGCGTTCTGCTATGGTTCACGTACGAGTTCTGAAATACCCACACAACATTCTGTTCACTAACCTGACCAACGACCTCTTCACCTACCTCCCTAAAACCTACAACGAAAGCAACTTCGTTTCTAACGTTCTGGAAGTTGAACTGAACGACGGCGAACTGTTCGTTCTGGCTTGCGAACTCATTAACAAAAAATGCTTCCAGGAAGGCAAAGAAAAAGCCCTGTACAAATCTAACAAAATCATTTACCACAACAAGCTCACTATATTCAAAGCTCCATTCTACGTTACCAGCAAAGACGTTAACACCGAATGCACCTGTAAATTCAAAAACAACAACTACAAAATCGTTCTGAAACCAAAATACGAAAAAAAAGTCATCCATGGCTGCAATTTTAGCAGCAACGTATCTAGCAAACACACTTTCACCGACTCTCTGGACATTAGCCTGGTTGACGACTCTGCTCACATTAGCTGCAATGTTCACCTCAGCGAACCAAAATACAACCACCTCGTTGGCCTGAACTGCCCAGGCGACATTATCCCAGACTGTTTCTTCCAGGTTTACCAGCCAGAAAGCGAAGAACTCGAACCATCGAATATTGTTTACCTGGACAGCCAGATCAACATCGGCGACATTGAATACTACGAAGACGCTGAAGGCGACGACAAAATTAAACTGTTCGGCATCGTTGGCTCTATCCCAAAAACGACCAGCTTCACTTGCATCTGCAAGAAGGACAAAAAATCTGCTTACATGACCGTTACTATCGACTCT

SEQ ID NO: 2 preferably includes a signal and anchor sequence.

SEQ ID NO: 9 corresponds to the codon harmonized DNA sequence (SEQ IDNO:2, shown in italics) and restriction enzyme sites shown in bold, 6×histidine codons shown as underlined, as affinity tags, and linkersequences, preferably used to facilitate cloning.

SEQ ID NO: 9 CATATGGCACACCATCATCATCATCATCCCGGGGGATCCGGTTCTGGTACCAATAACGACTTCTACAACCCATCGGCTCTCAACTCTGAAATCAGCGGCTTCATCGGCTACAAGTGCAACTTCAGCAACGAAGGCGTTCACAACCTGAAGCCAGACATGCGAGAACGACGCAGCATTTTCTGTAATATACACTCGTACTTCATCTACGACAAGATCCGTCTGATCATCCCAAAAAAAAGCTCGAGCCCAGAGTTCAAAATCCTGCCTGAAAAATGCTTCCAGAAAGTTTACACTGACTACGAGAACCGTGTTGAAACTGACATCTCGGAACTGGGCCTGATTGAATACGAAATCGAAGAAAACGACACCAATCCAAACTACAACGAACGCACCATCACGATCAGCCCATTCTCTCCAAAAGATATTGAATTCTTCTGCTTCTGCGACAACACTGAAAAGGTTATCAGCTCTATCGAAGGGCGTTCTGCTATGGTTCACGTACGAGTTCTGAAATACCCACACAACATTCTGTTCACTAACCTGACCAACGACCTCTTCACCTACCTCCCTAAAACCTACAACGAAAGCAACTTCGTTTCTAACGTTCTGGAAGTTGAACTGAACGACGGCGAACTGTTCGTTCTGGCTTGCGAACTCATTAACAAAAAATGCTTCCAGGAAGGCAAAGAAAAAGCCCTGTACAAATCTAACAAAATCATTTACCACAACAAGCTCACTATATTCAAAGCTCCATTCTACGTTACCAGCAAAGACGTTAACACCGAATGCACCTGTAAATTCAAAAACAACAACTACAAAATCGTTCTGAAACCAAAATACGAAAAAAAAGTCATCCATGGCTGCAATTTTAGCAGCAACGTATCTAGCAAACACACTTTCACCGACTCTCTGGACATTAGCCTGGTTGACGACTCTGCTCACATTAGCTGCAATGTTCACCTCAGCGAACCAAAATACAACCACCTCGTTGGCCTGAACTGCCCAGGCGACATTATCCCAGACTGTTTCTTCCAGGTTTACCAGCCAGAAAGCGAAGAACTCGAACCATCGAATATTGTTTACCTGGACAGCCAGATCAACATCGGCGACATTGAATACTACGAAGACGCTGAAGGCGACGACAAAATTAAACTGTTCGGCATCGTTGGCTCTATCCCAAAAACGACCAGCTTCACTTGCATCTGCAAGAAGGACAAAAAATCTGCTTACATGACCGTTACTATCGACTCT TGATAAGCGGCCGC

In other embodiments of the invention, the codon harmonized genecorresponds to the amino acid sequence corresponding to SEQ ID NO: 3,shown below, and the corresponding nucleic acid sequence, SEQ ID NO: 4.

SEQ ID NO: 3 KYNNAKVTVDTVCKRGFLIQMSGHLECKCENDLVINNEETCEEKVLKCDEKTVNKPCGDFSKCIKIDGNPVSYACKCNLGYDMVNNVCIPNECKNVTCGNGKCILDTSNPVKTGVCSCNIGKVPNVQDQNKCSKDGETKCSLKCLKENETCKAVDGIYKCDCKDGFIIDNESSICTAFSAYNILN SEQ ID NO: 4AAGTATAACAACGCCAAAGTTACTGTCGACACTGTATGTAAACGTGGTTTCCTGATTCAAATGAGCGGCCACCTCGAATGCAAATGCGAAAACGACCTGGTCCTGGTAAACGAAGAAACCTGCGAAGAAAAAGTTTTAAAATGCGATGAAAAGACTGTAAACAAACCGTGCGGTGACTTCTCGAAATGCATTAAAATTGACGGTAACCCTGTTTCTTATGCTTGCAAATGCAACCTCGGTTACGACATGGTAAACAACGTTTGCATTCCGAACGAATGCAAGAACGTAACTTGCGGCAATGGCAAATGCATTCTGGACACCTCGAACCCAGTTAAAACTGGTGTTTGTTCTTGCAACATTGGGAAAGTTCCTAACGTACAGGACCAGAACAAATGCTCTAAAGACGGTGAAACGAAATGTTCTCTGAAATGTCTGAAAGAAAACGAAACGTGCAAAGCTGTTGACGGTATTTACAAATGCGACTGCAAAGACGGTTTCATTATTGACAACGAATCGAGCATTTGCACTGCTTTCTCTGCTTACAACATTCTGAAC

SEQ ID NO:4 preferably includes a signal and anchor sequence.

SEQ ID NO: 8 corresponds to the codon harmonized DNA sequence (SEQ IDNO:4, shown in italics) and restriction enzyme sites shown in bold, 6×histidine codons shown as underlined, as affinity tags, and linkersequences, preferably used to facilitate cloning.

SEQ ID NO: 8 CATATGG CACACCATCATCATCATCATCCCGGGGGATCCGGTTCTGGTACCAAGTATAACAACGCCAAAGTTACTGTCGACACTGTATGTAAACGTGGTTTCCTGATTCAAATGAGCGGCCACCTCGAATGCAAATGCGAAAACGACCTGGTCCTGGTAAACGAAGAAACCTGCGAAGAAAAAGTTTTAAAATGCGATGAAAAGACTGTAAACAAACCGTGCGGTGACTTCTCGAAATGCATTAAAATTGACGGTAACCCTGTTTCTTATGCTTGCAAATGCAACCTCGGTTACGACATGGTAAACAACGTTTGCATTCCGAACGAATGCAAGAACGTAACTTGCGGCAATGGCAAATGCATTCTGGACACCTCGAACCCAGTTAAAACTGGTGTTTGTTCTTGCAACATTGGGAAAGTTCCTAACGTACAGGACCAGAACAAATGCTCTAAAGACGGTGAAACGAAATGTTCTCTGAAATGTCTGAAAGAAAACGAAACGTGCAAAGCTGTTGACGGTATTTACAAATGCGACTGCAAAGACGGTTTCATTATTGACAACGAATCGAGCATTTGCACTGCTTTCTCTGCTTACAACATTCTGAAC TGATAAGCGGCCGC

In other exemplary embodiments of the present invention, the codonharmonized gene corresponds to the amino acid sequence corresponding toSEQ ID NO: 5, shown below, and the corresponding nucleic acid sequence,SEQ ID NO: 6. SEQ ID NO: 5 shows the full length peptide sequence, wherethe signal peptide and the GPI anchor position are in bold.

MLKRQLANLLLVLSLLRGITHTQMAKGEVKYVPPEELNKDVSGFFGFKCNFSSKGVHNLEPILTEKRSLVCSIYSYFIYDKIKLTIPKKIPGSKFKMLPEKCFQTVYTNYEKRTEEKIENMGLVEYEVKEDDSNSEYTEKILTISPFNTKDVEFFCICDNSENVISNVKGRVALVQVNVLKYPHKITSINLTKEPYSYLPNQVDKTSFKSHKLDLELQDGELVVLACEKVDDKCFKKGKDTSPLSLYKSKKIVYHKNLSIFKAPVYVKSADVTAECSCNVDSTIYTLSLKPVYTKKLIHGCNFSSDKSTHNFTNHVDMAELGENAQITCSIELVDTSYNHLIGMSCPGEVLPECFFQVYQRESPELEPSKIVYLDAQLNIGNVEYFEDSKGENIVKIFGLVGSIPKTTSFTCICRKGKKIGYMSVK I AAGYF GFLAKIFILLIVLLLLYF*

SEQ ID NO:6 corresponds to the codon harmonized Pvs48/45 DNA sequenceconsisting of 1274 bases, not including the signal sequence and minusGPI anchor sequence shown in SEQ ID NO:5.

SEQ ID NO: 6 CATATGGCACACCATCATCATCATCATCCCGGGGGATCCGGTTCTGGTACCATGGCCAAGGGCGAGGTCAAATATGTGCCTCCAGAGGAGCTGAATAAGGATGTGAGCGGCTTTTTTGGGTTTAAGTGTAATTTTAGCAGCAAGGGCGTCCATAACCTCGAGCCTATTCTGACGGAGAAGCGAAGCCTGGTCTGTAGCATTTATAGCTATTTTATTTATGATAAGATTAAACTGACGATTCCTAAAAAAATTCCAGGCTCGAAGTTTAAGATGCTCCCAGAGAAGTGTTTTCAAACGGTGTATACTAATTATGAGAAGCGCACGGAGGAGAAGATTGAGAATATGGGCCTCGTGGAGTATGAGGTGAAGGAGGATGATAGCAACTCGGAGTATACGGAGAAGATTCTGACGATTAGCCCTTTTAACACGAAAGATGTGGAGTTTTTTTGTATTTGTGATAACTCGGAGAACGTCATTAGCAATGTGAAAGGCCGAGTGGCGCTCGTGCAAGTGAATGTGCTCAAGTATCCTCATAAGATTACGAGCATTAACCTGACGAAAGAGCCATATTCGTATCTGCCTAATCAAGTGGATAAAACGAGCTTTAAGTCGCATAAGCTCGATCTCGAGCTCCAAGATGGCGAGCTCGTGGTGCTCGCGTGTGAGAAGGTGGATGATAAGTGTTTTAAGAAAGGCAAAGATACTAGCCCACTGAGCCTCTATAAAAGCAAGAAGATTGTGTATCATAAAAACCTCAGCATTTTTAAGGCGCCTGTGTATGTGAAGAGCGCCGATGTGACGGCGGAGTGTAGCTGTAATGTGGATAGCACGATTTATACTCTCAGCCTCAAGCCTGTGTATACGAAGAAACTCATTCATGGGTGTAATTTTAGCTCGGATAAGAGCACGCATAATTTTACTAATCATGTGGATATGGCCGAGCTGGGGGAGAATGCGCAAATTACGTGTAGCATTGAGCTCGTGGATACGAGCTATAATCATCTCATTGGCATGAGCTGTCCTGGGGAGGTGCTGCCTGAGTGTTTTTTTCAAGTGTATCAACGAGAGAGCCCAGAGCTCGAGCCTAGCAAGATTGTCTATCTCGATGCCCAACTCAATATTGGCAATGTGGAGTATTTTGAGGATAGCAAAGGCGAGAATATTGTGAAGATTTTTGGGCTCGTGGGCAGCATTCCTAAGACGACGAGCTTTACGTGTATTTGTCGAAAAGGGAAAAAGATTGGGTATATGAGCGTGAAGTGATAAGCGGCCGC

In still other embodiments of the present invention, the codonharmonized gene corresponds to the nucleic acid sequence shown as SEQ IDNO: 10, where SEQ ID NO: 10 is a codon harmonized Pfs230 C-terminalmodified.

SEQ ID NO: 10 CATATG GCACACCATCATCATCATCATCCCGGGGGATCCGGTTCTGGTACCAAAGAATATGTTTGCGCTTCACCGACCAGCTGAAACCGACCGAATCTGGCCCAAAAGTTAAAAAATGCGAAGTTAAAGTTAACGAGCCGCTGATCAAAGTTAAAATCATCTGCCCGCTGAAAGGCAGCGTTGAAAAACTGTACGACAACATCGAATACGTTCCAAAAAAATCGCCGTACGTTGTTCTGACCAAAGAGGAAACTAAACTCAAGGAAAAACTGTTGTCGAAACTCATTTACGGCCTGCTGATCAGCCCTACGGTTAATGAAAAGGAGAACAACTTCAAAGAAGGCGTTATTGAATTCACTCTGCCTCCAGTGGTTCATAAGGCTACCGTGTTCTACTTCATCTGCGACAACAGCAAAACCGAAGACGACAATAAAAAAGGCAACCGTGGGATTGTTGAAGTGTACGTTGAACCGTACGGCAACAAAATTAACGGCTGCGCTTTTCTCGACGAAGACGAAGAAGAAGAAAAATACGGCAACCAGATTGAAGAAGACGAACACAACGAGAAGATCAAAATGAAAACCTTTTTCACGCAAAACATCTACAAAAAAAACAACATCTACCCGTGCTACATGAAACTGTACTCGGGCGACATCGGCGGCATTCTCTTCCCAAAGAACATCAAAAGCACCACGTGCTTCGAAGAGATGATCCCATACAACAAAGAAATCAAATGGAACAAAGAAAACAAATCTCTGGGCAATCTGGTTAACAACAGCGTTGTTTACAACAAAGAGATGAACGCTAAATACTTCAACGTTCAATACGTTCATATTCCAACCTCTTACAAAGACACCCTGAACCTGTTCTGCTCTATTATCCTGAAAGAAGAGGAATCTAACCTGATTAGCACTAGCTACCTGGTTTACGTTTCTATTAACGAAGAACTGAACTTCAGCCTCTTTGACTTCTACGAAAGCTTCGTTCCAATCAAAAAAACGATCCAGGTTGCTCAGAAGAACGTTAACAACAAAGAACACGACTACACCTGCGACTTCACGGACAAACTGGACAAAACGGTTCCAAGCACTGCTAACGGGAAGAAACTGTTCATCTGCCGTAAGCACCTGAAAGAATTCGACACCTTCACGCTGAAATGCAACGTTAACAAAACCCAGTACCCGAACATAGAGATCTTCCCAAAAACCCTGAAAGACAAAAAGGAAGTTCTGAAACTGGACCTCGACATCCAGTACCAGATGTTCTCTAAATTCTTCAAATTTAACACCCAAAACGCTAAGTACCTGAACCTGTACCCGTACTACCTGATTTTCCCGTTCAACCACATCGGCAAAAAAGAACTGAAAAACAACCCAACCTACAAAAACCACAAAGACGTGAAATACTTCGAGCAGAGCAGCGTTCTGAGCCCTCTGAGCTCGGCTGATTCTCTGGGGAAACTGCTGAACTTCCTGGACACTCAGGAGACGGTTTGCCTCACGGAAAAGATCCGTTACCTGAACCTGTCTATAAACGAGCTGGGCAGCGACAACAACACCTTCAGCGTTACCTTCCAAGTTCCGCCGTACATCGACATTAAGGAACCATTCTACTTCATGTTCGGCTGCAACAACAACAAAGGCGAAGGGAACATAGGCATTGTTGAACTGCTGATCAGCA AGCAG TGATAAGCGGCCGC

Here, the bold, underlined sequence refers to restriction enzyme sitesto facilitate cloning into an expression plasmid vector.

In other embodiments of the present invention, the codon harmonized genecorresponds to the nucleic acid sequence shown as SEQ ID NO: 11, whereSEQ ID NO: 11 corresponds to codon harmonized Pf230 A2B3.

SEQ ID NO: 11 CATATG GCACACCATCATCATCATCATCCCGGGGGATCCGGTTCTGGTACCCACGACTACACCTGCGACTTCACGGACAAACTGGACAAAACGGTTCCAAGCACTGCTAACGGGAAGAAACTGTTCATCTGCCGTAAGCACCTGAAAGAATTCGACACCTTCACGCTGAAATGCAACGTTAACAAAACCCAGTACCCGAACATAGAGATCTTCCCAAAAACCCTGAAAGACAAAAAGGAAGTTCTGAAACTGGACCTCGACATCCAGTACCAGATGTTCTCTAAATTCTTCAAATTTAACACCCAAAACGCTAAGTACCTGAACCTGTACCCGTACTACCTGATTTTCCCGTTCAACCACATCGGCAAAAAAGAACTGAAAAACAACCCAACCTACAAAAACCACAAAGACGTGAAATACTTCGAGCAGAGCAGCGTTCTGAGCCCTCTGAGCTCGGCTGATTCTCTGGGGAAACTGCTGAACTTCCTGGACACTCAGGAGACGGTTTGCCTGACGGAAAAGATCCGTTACCTGAACCTGTCTATAAACGAGCTGGGCAGCGACAACAACACCTTCAGCGTTACCTTCCAAGTTCCGCCGTACATCGACATTAAGGAACCATTCTACTTCATGTTCGGCTGCAACAACAACAAAGGCGAAGGGAACATAGGCATTGTTGAACTGCTGATCAGCAAGCAGGAAGAAAAGATTAAAGGCTGCAACTTTCACGAAAGCAAACTGGACTACTTTAACGAAAATATTAGCTCTGACACCCACGAATGCACCCTCCACGCTTACGAAAACGACATCATTGGCTTCAACTGCCTGGAAACTACTCACCCAAACGAGGTTGAGGTTGAAGTTGAAGACGCTGAAATCTACCTCCAGCCAGAGAACTGCTTCAACAACGTTTACAAAGGCCTCAACAGCGTTGACATTACTACTATCCTGAAAAACGCTCAGACCTACAACATCAACAACAAGAAAACCCCAACGTTCCTGAAAATTCCGCCGTACAACCTGCTGGAAGACGTCGAAATTTCTTGTCAGTGCACTATTAAACAGGTTGTTAAAAAAATCAAAGTTATTATCACGAAAAACGACACCGTTCTGCTGAAACGTGAAGTGCAGAGCGAGAGCACCCTGGACGACAAAATCTACAAATGCGAACACGAAAACTTCATTAACCCGCGTGTTAACAAAACCTTCGACGAAAACGTTGAATACACCTGCAACATCAAAATCGAGAACTTTTTCAACTACATTCAGATCTTCTGCCCGGCCAAAGACCTCGGCATTTACAAAAACATCCAGATGTACTACGACATTGTTAAACCGACCCGTGTTCCGCAGTTCAAAAAATTCAACAACGAAGAACTGCACAAACTGATTCCAAACAGCGAAATGCTGCACAAAACCAAAGAAATGCTGATTCTGTACAACGAAGAAAAAGTGGACCTCCTGCACTTCTACGTTTTTCTGCCGATCTACATCAAAGATATCTACGAATTTAACATCGTTTGCGACAACAGCAAAACCATGTGGAAAAACCAGCTGGGCGGCAAAGTTATCTACCACATTACTGTTAGCAAACGTGAGCAAAAAGTTAAAGGCTGCAGCTTCGACAACGAACACGCTCACATGTTCTCTTACAACAAAACTAACGTTAAAAACTGCATTATCGACGCTAAACCAAAAGACCTCATCGGCTTTGTTTGCCCTAGCGGCACGCTGAAACTGACCAACTGCTTCAAAGACGCTATCGTTCACACCAACCTGACCAACATTAACGGCATCCTCTACCTGAAAAACAACCTCGCTAATTTCACCTACAAACACCAGTTCAACTACATGGAAATCCCGGCTCTGATGGACAACGACATCAGCTTCAAATGCATCTGCGTTGACCTGAAAAAAAAAAAATACAACGTCAAAAGCCCGCTGGGCCCA TGATAAGCGGCCGC

Here, the bold, underlined sequence refers to restriction enzyme sitesto facilitate cloning into an expression plasmid vector.

In still other embodiments of the present invention, the codonharmonized gene corresponds to the nucleic acid sequence shown as SEQ IDNO: 12, where SEQ ID NO:12 corresponds to codon harmonized Pfs230 A4B5.

SEQ ID NO: 12 CATATG GCACACCATCATCATCATCATCCCGGGGGATCCGGTTCTGGTACCAACCGTCACGTTTGCGACTTTAGCAAAAACAACCTGATTGTTCCGGAAAGCCTGAAAAAAAAAGAAGAGCTCGGCGGCAACCCGGTTAACATTCACTGCTACGCTCTGCTGAAACCACTGGACACCCTGTACGTTAAATGCCCAACCAGCAAAGACAACTACGAAGCTGCTAAAGTTAATATCAGCGAAAATGATAACGAATACGAGCTGCAGGTTATCAGCCTGATAGAAAAACGTTTCCACAACTTCGAGACGCTGGAATCGAAGAAACCAGGCAACGGCGACGTTGTTGTTCACAACGGCGTTGTTGACACTGGCCCAGTTCTGGACAATTCTACCTTCGAAAAATACTTCAAAAACATCAAAATCAAACCGGACAAATTCTTCGAGAAAGTTATCAACGAATACGACGACACTGAAGAAGAAAAAGACCTGGAATCTATCCTGCCAGGGGCTATTGTTTCTCCAATGAAAGTTCTGAAAAAAAAGGACCCATTCACCAGCTACGCTGCTTTCGTTGTTCCGCCGATTGTTCCTAAAGACCTGCACTTCAAAGTTGAATGCAACAACACCGAATACAAAGACGAAAACCAGTACATCTCTGGCTACAACGGCATCATCCACATTGACATCAGCAACTCTAACCGCAAAATTAACGGCTGCGACTTTAGCACTAATAACTCTAGCATTCTGACCTCGTCTGTTAAACTGGTTAACGGCGAAACTAAAAACTGCGAAATCAACATCAACAACAACGAAGTTTTCGGCATAATCTGCGACAACGAAACCAACCTGGACCCGGAAAAATGCTTCCACGAAATCTACTCTAAAGACAACAAAACTGTTAAAAAATTCCGAGAAGTTATCCCAAACATCGACATCTTTAGCCTGCACAACAGCAACAAGAAAAAAGTTGCTTACGCTAAAGTTCCACTGGACTACATTAACAAACTGCTGTTCAGCTGCAGCTGCAAAACCAGCCACACTAACACCATCGGCACGATGAAAGTTACTCTCAACAAAGACGAAAAAGAAGAAGAAGACTTCAAAACCGCTCAGGGCATTAAACACAACAACGTTCACCTGTGCAACTTTTTCGACAACCCAGAACTGACCTTCGACAACAACAAAATCGTTCTGTGCAAAATAGACGCTGAATTATTTAGCGAAGTTATTATCCAGCTGCCGATCTTCGGCACCAAGAACGTTGAAGAAGGCGTTCAGAACGAAGAATACAAAAAATTCAGCCTGAAACCGAGCCTGGTTTCGACGACAATAACAACGACATTAAAGTTATCGGCAAAGAAAAAAACGAAGTTAGCATTTCTCTGGCTCTCAAAGGGGTTTACGGCAACCGAATTTTCACTTTCGACAAAAACGGCAAAAAAGGCGAAGGCATTTCTTTCTTCATCCCACCGATCAAACAGGACACCGACCTGAAATTCATCATTAACGAAACCATCGACAACAGCAACATTAAACAGCGTGGCCTGATCTACATTTTCGTTCGCAAAAACGTTAGCGAAAACAGCTTCAAACTGTGCGACTTTACCACCGGCTCGACTAGCCTGATGGAACTGAACTCTCAGGTTAAAGAAAAAAAGTGTACTGTTAAAATTAAAAAAGGCGACATTTTCGGCCTCAAATGCCCAAAAGGCTTCGCTATCTTCCCGCAGGCTTGCTTCTCTAACGTTCTGCTGGAATACTACAAATCTGACTACGAAGACTCTGAACACATTAACTACTACATTCACAAAGACAAAAAATACAACCTGAAACCAAAAGACGTTATTGAACTGATGGACGAAAACTTCCGTGAACTGCAGAACATCCAGCAGTACACCGGCATCAGCAACATTACCGACGTGCTGCACTTTAAAAACTTCAACCTGGGCAACCTCCCGCTGAACTTCAAAAACCACTACAGCACCGCTTACGCTAAAGTTCCGGACACGTTCAACAGCATTATTAATTTTAGCTGCAACTGCTACAACCCGGAAAAACACGTTTACGGCACTATGCAGGTTGAGAGCGACAAC TGATAAGCGGCCGC

Here, the bold, underlined sequence refers to restriction enzyme sitesto facilitate cloning into an expression plasmid vector.

In other embodiments of the present invention, the codon harmonized genecorresponds to the nucleic acid sequence shown as SEQ ID NO: 13, whereSEQ ID NO: 13 corresponds to codon harmonized Pfs230 A6B7.

SEQ ID NO: 13 CATATG GCACACCATCATCATCATCATCCCGGGGGATCCGGTTCTGGTACCAACGAACACATCTGCGACTACGAAAAAAACGAAAGCTTAATCAGCACCCTGCCAAACGACACCAAAAAAATCCAGAAATCTATATGCAAAATTAACGCTAAAGCTCTGGACGTTGTTACCATTAAATGCCCACACACCAAAAACTTCACGCCAAAAGACTACTTCCCAAACAGCAGCCTGATCACTAACGACAAAAAAATTGTGATTACTTTCGACAAGAAAAACTTCGTTACTTACATCGACCCAACCAAAAAAACCTTCAGCCTCAAAGACATCTACATCCAGTCTTTCTACGGCGTTAGCCTCGACCACCTCAACCAGATCAAAAAAATCCACGAAGAATGGGACGACGTTCACCTGTTCTACCCACCACACAACGTTCTGCACAACGTTGTTCTCAACAACCACATCGTCAATCTGAGCAGCGCTCTGGAAGGCGTCCTGTTCATGAAAAGCAAAGTTACTGGCGACGAAACCGCTACCAAAAAAAATACTACCCTCCCGACTGACGGCGTTAGCTCTATTCTGATTCCGCCGTACGTTAAGGAAGACATCACCTTCCACCTCTTCTGCGGGAAAAGCACCACCAAAAAACCGAATAAAAAGAATACCAGCCTCGCTCTCATTCACATCCACATCAGCAGCAATCGTAACATTATTCACGGCTGCGACTTTCTGTACCTGGAAAACCAGACCAACGACGCTATTTCTAACAACAACAACAACAGCTACAGCATCTTCACCCACAACAAAAACACCGAGAACAACCTCATCTGCGACATCAGCCTGATTCCGAAAACTGTTATCGGCATTAAATGCCCAAACAAAAAACTGAACCCGCAGACCTGCTTCGACGAAGTGTACTACGTTAAACAGGAAGACGTTCCATCGAAAACTATCACCGCTGACAAATACAACACCTTCTCTAAAGATAAAATCGGCAACATCCTGAAAAACGCTATAAGCATTAACAACCCGGACGAAAAGGACAACACCTACACTTACCTGATCCTGCCGGAAAAATTCGAAGAAGAACTGATAGACACGAAAAAAGTTCTGGCTTGCACCTGCGACAACAAATACATCATCCACATGAAAATCGAAAAATCTACCATGGACAAAATCAAAATCGACGAAAAAAAAACCATTGGCAAAGACATCTGCAAATACGACGTTACTACTAAAGTTGCTACTTGCGAAATTATTGACACCATTGACTCGAGCGTTCTGAAAGAACACCACACCGTTCACTACAGCATTACCCTGAGCCGTTGGGACAAACTCATTATTAAATACCCGACCAACGAGAAAACCCACTTTGAAAACTTCTTCGTTAACCCATTCAACCTGAAAGACAAAGTTCTGTACAACTACAACAAACCGATCAACATCGAACACATACTGCCGGGCGCCATTACCACCGACATCTACGACACGCGTACCAAAATTAAACAGTACATCCTGCGTATTCCGCCGTACGTTCACAAAGACATCCACTTTAGCCTGGAATTCAATAACTCGCTCTCTCTGACCAAACAGAACCAGAACATTATTTACGGCAACGTTGCCAAAATTTTCATTCACATCAACCAGGGCTACAAAGAAATTCACGGCTGCGACTTTACCGGCAAATACTCGCACCTGTTCACCTACAGCAAAAAACCACTGCCGAACGACGACGACATCTGCAACGTTACTATCGGCAACAACACCTTTAGCGGCTTCGCTTGTCTGTCGCACTTCGAACTGAAACCGAACAATTGTTTTAGCAGCGTTTACGACTACAACGAAGCCAACAAAGTTAAAAAACTGTTTGACCTCTCGACCAAAGTTGAACTGGATCACATAAAACAGAACACTAGCGGCTACACCCTCAGCTACATTATTTTCAACAAAGAATCGACCAAACTCAAATTTAGCTGCACCTGTAGCTCGAATTACAGCAACTACACTATCCGAATAACC TTCGACCCATGATAAGCGGCCGC

Here, the bold, underlined sequence refers to restriction enzyme sitesto facilitate cloning into an expression plasmid vector.

The compositions of the invention are designed for expression in a host.In preferred embodiments, a host is E. coli or an E. coli derivative.The DNA encoding the desired recombinant protein can be introduced intoa host cell in any suitable form including, the fragment alone, alinearized plasmid, a circular plasmid, a plasmid capable ofreplication, an episome, RNA, etc. Preferably, the gene is contained ina plasmid. In a particularly preferred embodiment, the plasmid is anexpression vector. Individual expression vectors capable of expressingthe genetic material can be produced using standard recombinanttechniques. Please see e.g., Maniatis et al., 1985 Molecular Cloning: ALaboratory Manual or DNA Cloning, Vol. I and II (D. N. Glover, ed.,1985) for general cloning methods

Accordingly, the present invention contemplates host cells transformedwith a vector as described herein. For example, the vector may comprisea codon harmonized Pfs48/45 sequence suitable for expression in a cell,a codon harmonized Pvs48/45 sequence suitable for expression in a cell,a codon harmonized Pfs25 sequence suitable for expression in a cell.

The one or more Plasmodium pre-fertilization or post-fertilizationantigens, for example Plasmodium falciparum or Plasmodium vivaxpre-fertilization or post-fertilization antigens can be used singly, orin combinations. For example, a combination may comprise antigensderived from different Plasmodium species that will be capable ofblocking the infection and/or transmission of more than one Plasmodiumspecies. For example, an immunogenic composition comprising Pfs48/45 andPvs48/45 will be capable of blocking the transmission of both P.falciparum and P. vivax.

METHODS OF THE INVENTION

The present invention describes an approach that harmonizes codon usagefrequency of the target gene with those of the expression host forheterologous expression of protein. Basic studies on regulation ofprotein expression have shown that synonymous codon substitutions frominfrequent to frequent usage in regions where mRNA translation occursrelatively slowly can be detrimental to protein expression and stability[17]. On the other hand, codon substitutions introducing rare codons inthe regions containing high frequency codons can lead to erroneousprotein conformation [18]. Taking these concepts into account, analgorithm termed “codon harmonization” [19] was developed wheresynonymous codons from E. coli were selected that closely resemble thecodon usage of native Pfs48/45 gene, including regions coding ‘link/end’segments of proteins in P. falciparum. This approach harmoniously mimicsthe translation rate of protein expression in native host by allowingthe translation machinery to pause at exactly the same positions in E.coli as in P. falciparum and yield expression of correctly foldedprotein, for example, Pfs48/45 in E. coli.

The present invention describes for the first time the efficient andsuccessful expression of a pre-fertilization antigen, and in particular,full length TBV candidate Pfs48/45, in high yields and appropriateconformation. The recombinant Pfs48/45 elicits potent malariatransmission blocking antibodies in mice and non human primates (Olivebaboons, Papio anubis) and indicates the development of a malaria TBVbased on the CH-rPfs48/45 antigen.

In certain preferred embodiments of the invention, the pre-fertilizationor post-fertilization antigens can be used to develop malariatransmission-blocking immunogenic compositions. Accordingly, theinvention features methods of blocking transmission of Plasmodiumfalciparum or Plasmodium vivax in a subject comprising administering toa subject an immunogenic composition comprising one or more Plasmodiumfalciparum or Plasmodium vivax pre-fertilization or post-fertilizationantigens, thereby blocking transmission of Plasmodium falciparum orPlasmodium vivax in the subject.

In certain embodiments, it may be desirable to measure transmission, forexample, by measuring the reduction of mosquito oocysts by sera orplasma from a subject treated with the immunogenic composition comparedto a control subject.

Accordingly, pre-immune sera from the treated subject and the controlsubject are used as a measure of 100% transmission for the correspondingtest sera.

The invention also features methods of immunizing a subject againstPlasmodium falciparum or Plasmodium vivax comprising administering to asubject an immunogenic composition comprising one or more Plasmodiumfalciparum or Plasmodium vivax pre-fertilization or post-fertilizationantigens, and thereby immunizing the subject against Plasmodiumfalciparum or Plasmodium vivax.

The immunogenic compositions of the invention are also preferably usedin methods for treating or preventing malaria in a subject comprisingadministering to a subject an immunogenic composition comprising one ormore Plasmodium falciparum or Plasmodium vivax pre-fertilization orpost-fertilization antigens, thereby preventing malaria in the subject.

In preferred embodiments, the pre-fertilization or post-fertilizationantigens can be classified as Plasmodium falciparum or Plasmodium vivaxsurface antigens. In certain preferred embodiments, the surface antigensare gametocyte or gamete surface antigens. Exemplary gametocyte orgamete surface antigens are selected from the group consisting ofPfs48/45, Pfs230, Pvs48/45 and Pvs230. In other embodiments, the surfaceantigens are midgut parasite surface antigens. Exemplary midgut parasitesurface antigens are selected from the group consisting of Pfs25, Pfs28,Pvs25 and Pvs28.

Preferably, the one or more pre-fertilization or post-fertilizationantigens are selected from the group consisting of Pfs48/45, Pfs 230,Pfs25, Pvs48/45, Pvs 230 and Pvs25. In particular, in certainembodiments, the pre-fertilization antigen is Pfs48/45. In otherembodiments, the post-fertilization antigen is Pfs25.

As described herein, codon harmonized genes are preferably employed inthe methods of the invention, where, generally, a wild type nucleic acidsequence encoding a polypeptide is modified to enhance expression andaccumulation of the polypeptide in the host cell by harmonizingsynonymous codon usage frequency between the foreign DNA and the hostcell DNA. Accordingly, in certain preferred embodiments, of theinvention, one or more pre-fertilization antigens or post-fertilizationantigens is derived from a codon harmonized gene.

Codon harmonized genes are described herein, and in particularembodiments the modified (codon harmonized) nucleic acid sequence and/orthe encoded polypeptide are shown in SEQ ID NOs 1-SEQ ID NO: 13.

Administration

The immunogenic compositions of the present invention can beadministered to a subject by different routes such as subcutaneous,intradermal, intramuscular, intravenous and transdermal delivery.Suitable dosing regimens are preferably determined taking into accountfactors well known in the art including age, weight, sex and medicalcondition of the subject; the route of administration; the desiredeffect; and the particular composition.

The course of the immunization may be followed by assays for activated Tcells produced, skin-test reactivity, antibody formation or otherindicators of an immune response to a malarial strain.

Dosage form, such as injectable preparations (solutions, suspensions,emulsions, solids to be dissolved when used, etc.), tablets, capsules,granules, powders, liquids, liposome inclusions, ointments, gels,external powders, sprays, inhalation powders, eye drops, eye ointments,and the like, can be used appropriately depending on the administrationmethod. Pharmaceutical formulations are generally known in the art andare described, for example, in Chapter 25.2 of Comprehensive MedicinalChemistry, Volume 5, Editor Hansch et al, Pergamon Press 1990.

Pharmaceutically acceptable carriers which can be used in the presentinvention include, but are not limited to, an excipient, a stabilizer, abinder, a lubricant, a colorant, a disintegrant, a buffer, an isotonicagent, a preservative, an anesthetic, and the like which are commonlyused in a medical field.

Immunogenic compositions are administered in immunologically effectiveamounts. An immunologically effective amount is one that stimulates theimmune system of the subject to establish a level of immunologicalresponse sufficient to reduce parasite density and disease burden causedby infection with the pathogen, and/or sufficient to block thetransmission of the pathogen in a subject.

A dose of the immunogenic composition may, in certain preferredembodiments, consist of the range of 1 μg to 1.0 mg total protein. Incertain preferred embodiments, the composition is administered in aconcentration between 1-100 μg. However, one may prefer to adjust dosagebased on the amount of antigen delivered. In either case these rangesare guidelines. More precise dosages should be determined by assessingthe immunogenicity of the composition so that an immunologicallyeffective dose is delivered. The immunogenic composition can be used inmulti-dose formats.

The timing of doses depends upon factors well known in the art. Afterthe initial administration one or more booster doses may subsequently beadministered to maintain antibody titers, e.g., the compositions of thepresent invention can be administered one time or serially over thecourse of a period of days, weeks, months and or years. An example of adosing regime would be day 1 an additional booster doses at distanttimes as needed. The booster doses may be administered at 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more weeks afterthe primary immunization. In preferred embodiments, the booster dosesare administered at 4 weeks. In other preferred embodiments, the boosterdoses are administered at 12 weeks.

In other cases, the immunogenic compositions are administered after,before or at the same time as treatment with an additional agent, suchas an agent to treat or prevent malaria.

As used herein the subject that would benefit from the immunogeniccompositions described herein include any host that can benefit fromprotection against malarial infection. Preferably, a subject can respondto inoculation with the immunogenic compositions of the presentinvention by generating an immune response. The immune response can becompletely or partially protective against symptoms caused by infectionwith a pathogen such as Plasmodium falciparum, or can block transmissionof the pathogen by Anopheles mosquitoes. In a preferred embodiment, thesubject is a human. In another embodiment, the subject is a non-humanprimate.

Formulations

The immunogenic compositions of the present invention can be used toimmunize mammals including humans against infection and/or transmissionof malaria parasite, or to treat humans post-infection, or to boost apathogen-neutralizing immune response in a human afflicted withinfection of malaria parasite.

The immunogenic compositions of the present invention can be formulatedaccording to methods known and used in the art. Guidelines forpharmaceutical administration in general are provided in, for example,Modern Vaccinology, Ed. Kurstak, Plenum Med. Co. 1994; Remington'sPharmaceutical Sciences 18th Edition, Ed. Gennaro, Mack Publishing,1990; and Modern Pharmaceutics 2nd Edition, Eds. Banker and Rhodes,Marcel Dekker, Inc., 1990. Immunogenic compositions of the presentinvention can be prepared as various salts. Pharmaceutically acceptablesalts (in the form of water- or oil-soluble or dispersible products)include conventional non-toxic salts or the quaternary ammonium saltsthat are formed, e.g., from inorganic or organic acids or bases.Examples of such salts include acid addition salts such as acetate,adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate,butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate;and base salts such as ammonium salts, alkali metal salts such as sodiumand potassium salts, alkaline earth metal salts such as calcium andmagnesium salts, salts with organic bases such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such ashistidine, arginine and lysine.

Adjuvants are almost always required to enhance and/or properly directthe immune response to a given antigen. An ideal adjuvant should besafe, stable with long shelf life, biodegradable, inexpensive andpromote an appropriate immune response while itself beingimmunologically inert. Adjuvants affect processes including antigenpresentation, antigen uptake and selective targeting of antigens thuscritically determining the magnitude and type of the immune responses[34-36]. While the mechanisms by which different adjuvants result indifferent outcomes remain a “black box”, studies strive for developing avaccine that can provide maximum efficacy with ease of delivery in asfewer doses as possible. It must be kept in mind that an adjuvant is notthe active component in a vaccine and immunization; outcomes can varygreatly from one adjuvant to another when used in combination with thesame vaccine antigen. Any given adjuvant-vaccine combination has to beevaluated on a case-by-case basis for safety, reactogenicity andefficacy in pre clinical trials. Ultimately, safety considerationsoutweigh any anticipated benefit and need to be evaluated for thedevelopment of a plan leading to human clinical trial [37].

Although aluminum compounds have been used for human vaccines since1926, the need for developing new and more effective adjuvants has beenfelt increasingly for many other subunit and DNA vaccines, especiallysince the alum salts are relatively poor adjuvants [38]. Otherexperimental adjuvants used in a limited number of studies in humansinclude Quil A-derived saponin QS-21, bacterial and fungal derivedmoieties (e.g. muramyl dipeptide, monophosphoryl lipid A, CpG etc.),Water-in-oil emulsions (e.g., MF59, FIA, Montanide), particulatedelivery systems (e:g., VLPs, liposomes, microspheres, ISCOM).

In certain preferred embodiments, the immunogenic compositions areformulated with an aluminum adjuvant. Aluminum based adjuvants arecommonly used in the art and include aluminum phosphate, aluminumhydroxide, aluminum hydroxy-phosphate, and amorphous aluminumhydroxyphosphate sulfate. Trade names of aluminum adjuvants in commonuse include ADJUPHOS, ALHYDROGEL, (both from Superfos Biosector a/s,DK-2950 Vedbaek, Denmark).

Non-aluminum adjuvants can also be used. Non-aluminum adjuvants include,but are not limited to, QS21, Lipid-A, Iscomatrix., and derivatives orvariants thereof, Freund's complete or incomplete adjuvant, neutralliposomes, liposomes containing vaccine and cytokines or chemokines.

Emulsions of Montenide ISA 51 (a mineral oil adjuvant) and ISA 720(oil-based non mineral oil) have been used in human clinical trials. Areview of clinical trials (25 trials representing more than 4000patients and 40,000 injections for Montanide ISA 51 and various trialsrepresenting 500 patients and 1500 injections for Montanide ISA 720) hasrevealed their general safety and strong adjuvant effect with mild tomoderate local reactions.

In certain preferred embodiments of the invention, the method of theinvention further comprises administering an adjuvant. In certainexamples, the adjuvant is selected a water-in-oil emulsion. In otherexamples, the adjuvant is Aluminum hydroxide. However, any adjuvant thatis suitable for administration with the immunogenic composition in themethods of the present invention can be suitably used.

Kits

Also included within the scope of the present invention are kitssuitable for providing compositions of the immunogenic compositions asdescribed herein.

For example, in such a kit one vial can comprise the immunogeniccomposition comprising one or more Plasmodium falciparum or Plasmodiumvivax pre-fertilization or post-fertilization antigens, and instructionsfor use in reducing transmission of Plasmodium falciparum or Plasmodiumvivax.

In another example, a kit can preferably comprise one or more Plasmodiumfalciparum or Plasmodium vivax pre-fertilization or post-fertilizationantigens, and instructions for use in preventing malaria.

Preferably, the one or more pre-fertilization or post-fertilizationantigens are selected from the group consisting of Pfs48/45, Pfs230, Pfs28, Pfs25, Pvs48/45, Pvs 230, Pvs 28, and Pvs25. In exemplaryembodiments, the one or more pre-fertilization or post-fertilizationantigens is derived from a codon harmonized gene.

Preferably, the kit will contain instructions for using the composition.Such instructions can be in the form of printed, electronic, visual, andor audio instructions.

EXAMPLES Example 1 Expression and Purification of Correctly FoldedPfs48/45

Generally speaking it is often difficult to express P. falciparumproteins in a heterologous expression system and such problems arelargely due to high A/T content giving rise to changes in codon usagefrequencies. It is now increasingly being realized that synonymous codonsubstitutions are not always silent and changes in the frequency ofcodon usage affects protein structure and function. Differences insynonymous codon usage between expression and natural hosts greatlyaffect expression and stability of proteins. These codon differenceshave even greater consequences if they occur infrequently and within thedomains that encode polypeptide regions of higher ordered structure. Thepresence of infrequently used codons is believed to cause ribosomepausing leading to incorrect folding and premature termination ofprotein during translation. An algorithm that takes into account knownrelationships between codon usage frequencies and secondary proteinstructure (designated codon harmonization algorithm) [25] has beendeveloped to identify regions of slowly translated mRNA that areputatively associated with ‘link/end’ polypeptide segments contributingto higher ordered structures in a protein. Thus modification of those‘link/end’ region codons and other codons to match the frequency in theexpression host can counter non-natural ribosomal pausing and allow forco-translational folding, of proteins as synthesis continues [25]. Byallowing the translation machinery to slow or pause at the same positionin the transcript in the expression host as in the natural host (i.e.Plasmodium), the secondary structures in the nascent proteins are formedat comparable rates and thus lead to proper conformational folding andsolubility of the expressed protein.

The native sequence of Pfs48/45 is represented by NCBI accession numberAF356146, corresponding to SEQ ID NO: 7.

Here, the native sequence of Pfs 48/45 as represented by SEQ ID NO: 7,lacking N-terminal signal sequence (amino acid residues 1-27) andC-terminal anchor (amino acid residues 428-448) was converted to ‘codonharmonized’ sequence designed for expression in E. coli. The amino acidsequence reported under AF356146 differs from that of Z22145 (NF54) byC32Y, K33N, T72N, K253N and N254K substitutions. The A/T contents ofPfs48/45 sequence before and after codon harmonization were 75% and 56%,respectively. The codon harmonized Pfs48/45 sequence containing asequence tag coding for 6× histidines at the 5′ end was synthesized andcloned into the pET (K-) expression vector [19] and expressed in BL21(DE3) cells [FIG. 1 a]. Initial attempts to induce the cells with IPTGindicated that the expressed protein might negatively impact the growthof E. coli. To overcome the toxicity of expressed protein, theexpression strategy was modified to slow down protein expression bygrowing the cells at 30° C. in Luria-Bertani growth medium containing 1%glucose and induction with 0.1 mM IPTG for 3 h, which resulted in highlyefficient induction of protein in the cell lysate at 50 kDa [FIG. 1 b,left panel, encircled]. The cell lysate when treated with β-ME to reducethe disulphide bonds in the protein, showed slower electrophoreticmobility of the induced Pfs48/45 protein [FIG. 1 b, right panel,encircled]. A similar observation was made with the native form of theprotein on the gametocyte surface [22,23].

Western blot analysis of cell lysates either untreated or aftertreatment with 0.5% sarcosyl revealed that the recombinant protein wasinsoluble in the absence of sarcosyl detergent (data not shown), andhence the treatment with ionic detergent was required to facilitateextraction of the protein from the pellet. To selectively enrichexpressed Pfs48/45, the lysate was first treated with non-ionicdetergent Tween-80 to remove any soluble bacterial proteins followed bytreatment with 0.5% sarcosyl in PBS [FIG. 1 c]. This sequentialdetergent extraction resulted in partial solubilization of the expressedprotein which could be further purified using Ni2+-NTA-Agarose beads(QIAGEN) by elution using imidazole (1 M), yielding ˜3 mg purifiedprotein/g of wet cell pellet [FIGS. 1 d, e]. The yield of purifiedCH-rPfs48/45 in 7 independent purifications varied between 15 and 25 mgper liter of induced culture. The conformational characterization of theexpressed protein designated CH-rPfs48/45 was achieved using a mAbIIC5-B10 [22], which recognizes a conformational reduction-sensitiveepitope in the parasite derived native Pfs48/45. The mAb recognized thenon-reduced form of CH-rPfs48/45 yielding 3 immunoreactive bands at ˜50kDa of the gel [FIG. 1 f, lane 1]. In addition to these monomeric forms,purified CH-rPfs48/45 protein preparations consistently revealed thepresence of a higher molecular weight band at ˜98 kDa, presumed to be adimer. When a similar Western blot analysis was carried out withCH-rPfs48/45 after reduction prior to SDS-PAGE, the mAb ratherunpredictably showed recognition of a single band around 65-68 kDa [FIG.1 f, lane 2]. Previous biosynthetic metabolic labeling studies hadestablished that non-reduced Pfs48/45 immunoprecipitated by the mAbIIC5B10 (a doublet migrating in the region of 45 and 48 kDa) coalescesinto a single protein band of ˜68 kDa when analyzed by SDS-PAGE afterreduction [23]. On the other hand, the same mAb recognized only thenon-reduced form of native Pfs48/45 in the gametocytes and gametes inWestern blot analysis [24]. While the reasons for the totally unexpectedrecognition of reduced form of CH-rPfs48/45 are not so obvious, apossible interpretation may be that the functional target epitope ofblocking antibodies might be conformationally more stable in theCH-rPfs48/45, and therefore not affected by the reduction conditionsemployed.

Example 2 Evaluation of Functional Immunogenicity of CH-rPfs48/45 inMice in Different Adjuvant Formulations

The immunogenicity of CH-rPfs48/45 was assessed in three differentadjuvant formulations: CFA, water-in-oil emulsion using Montanide ISA-51and Aluminium hydroxide. FIG. 2 a shows the IgG titer 2 weeks after the2nd boost in CFA group and 2 weeks after the 3rd boost in Alum andMontanide ISA-51 groups. This formulation resulted in the highestantibody titer ranging from 3×10⁵ to >1×10⁶ [FIG. 2 a, top panel].However, all mice immunized with CH-rPfs48/45 in the other two adjuvantformulations also showed high antibody titer, the range being ˜70,000 tomore than 100,000 in both Montanide ISA-51 and Alum formulations [FIG. 2a, middle and bottom panels]. Sera for IgG isotype distribution was alsotested and all four subtypes (IgG1, IgG2a, IgG2b, IgG3) were more orless equally represented in sera from mice immunized with CFAformulation, [FIG. 2 b, top panel]. On the other hand, IgG1 and to alesser extent IgG2 were the dominant subtype in Montanide ISA-51formulation [FIG. 2 b, middle panel]. The overwhelming presence of IgG1and negligible amount of IgG3 were noticed in the Alum formulation [FIG.2 b, bottom panel].

Individual mouse sera were also tested for their ability to recognizethe native Pfs48/45 protein in gametocyte extracts in both reduced andnon-reduced form in Western blot analysis. Polyclonal antisera againstCH-rPfs48/45 recognized a combination of reduction-sensitive as well asreduction-insensitive epitopes in Pfs48/45 in the parasites. Sera frommice immunized with CFA and ISA-51 formulations recognized the nativenon reduced protein (48/45 kDa), however, the sera from mice immunizedwith alum formulation revealed recognition of 48/45 kDa protein and alsoa protein ˜98 kDa [FIG. 3 a, left panel]. The reduced form of the nativeprotein was recognized by all of the aforementioned sera at ˜64 kDa[FIG. 3 a, right panel]. As previously demonstrated [24] the mAb IIC5B10reacted only with nonreduced native parasite antigen. The ability of theanti-CH-rPfs48/45 sera to recognize the native form of the protein wasfurther tested by live immunofluorescence assay (IFA) using liveextracellular gametes. FIG. 3 b shows an example of strong gametesurface reactivity typical of that observed for sera from mice immunizedwith CH-rPfs48/45 in all three adjuvant groups.

To assess the functionality of the immunized sera, they were tested fortransmission blocking activity in mosquitoes in membrane feeding assays(MFA) [10]. All the mice sera in the three adjuvant groups showed astrong (>98% reduction in the number of oocysts) transmission blockingactivity at 1:2 dilution [Table 1, shown below] as compared tocorresponding pre-immune sera.

TABLE 1 Serum dilution Animal no. 1:2 1:4 1:8 CFA 1 100.0 (0/20)  89.6(8/20)  76.8 (18/33) 2  97.5 (14/41) 88.0 (7/16)  79.3 (24/38) 3 98.3(7/28) 90.2 (4/15)  72.6 (16/34) 4  94.3 (12/22) 90.5 (9/26)  83.5(21/42) 5 98.0 (9/30) 93.9 (6/20)  84.6 (15/28) ISA 51 6 96.1 (5/15)88.0 (16/23) 76.8 (12/19) 7 98.4 (2/12) 89.9 (11/20) 74.9 (10/17) 8 96.4(7/18) 92.5 (12/22) 69.7 (16/22) 9 96.2 (7/15) 90.6 (15/25) 72.6 (11/18)10  95.1 (10/20) 86.2 (10/17) 79.1 (12/19) Alum 11  93.5 (11/24) 86.2(14/30) 54.9 (21/29) 12 95.3 (9/26) 78.6 (16/24) 58.3 (14/20) 13  96.6(12/31) 89.9 (6/15)  55.4 (15/22) 14 94.5 (7/16) 89.2 (9/17)  70.5(16/26) 15 95.2 (5/16) 91.6 (6/16)  61.4 (16/23)

Table 1 shows MFA with sera from mice immunized with CH-rPfs48/45formulated in CFA, Montanide ISA-51 or Alum. Individual mouse sera weretested for transmission blocking activity with respect to correspondingpooled pre-immune sera. Data are represented as percent transmissionblocking activity (reduction in the number of oocysts per mosquitomidgut). Numbers within parenthesis represent total number of infectedmosquitoes/total number of mosquitoes dissected for each feed.

Geometric mean oocyst numbers per midgut in the presence of pooledpre-immune sera of mice immunized in various adjuvant formulations andtested at different dilutions ranged between 10.4 and 16.7. The decreasein oocyst number/midgut by each immune sera was significant (P<0.02,Mann-Whitney test).

The transmission blocking effect was dependent upon the antibody dose asrevealed by a gradual decrease with increasing sera dilutions. Sera frommice immunized with CH-rPfs48/45 vaccine in CFA and Montanide ISA-51formulations as compared to Alum appeared to be relatively more potentblockers as apparent from the stronger transmission blocking activitiesat 1:8 dilution of sera in MFA.

Example 3 Evaluation of Functional Immunogenicity of CH-rPfs48/45 in NonHuman Primates (Olive Baboons)

Backed by strong functional immunogenicity of CH-rPfs48/45 in threedifferent adjuvant formulations in mice, the CH-rPfs48/45 vaccine wasnext evaluated in nonhuman primates (Papio anubis, Olive baboons). Thevaccine trial in baboons was approved by the institutional andscientific review committee of the Institute of Primate Research with aprotocol #19/10/2007.

A group of 5 baboons (ranging 7.6 to 12.2 kg in body weight) wereimmunized with 50 μg of CH-rPfs48/45 in Montanide ISA-51, water-in-oilemulsion. Our choice of the adjuvant for these studies was dictated, inpart, by the fact that the adjuvant has already been in use as aninvestigational adjuvant in clinical trials in humans [25] and that theCH-rPfs48/45 vaccine formulated in Montanide ISA-51 was stronglyeffective in murine immunization studies (above). The vaccine, dose,route and schedules selected were based on experience with numerousother malaria vaccine trials in nonhuman primates [26,27,28]. Here, thevaccine was delivered through the intramuscular route (quadriceps, twosites) and boosted twice with the same dose of protein at 4 and 12 weekspost primary immunization [FIG. 4 a].

To evaluate the immunogenicity of CH-rPfs48/45 in baboons, blood sampleswere collected from the immunized animals and assessed by ELISA. All,except one animal (Pan 3275), responded strongly after the primaryimmunization showing more than 8×104 IgG titers 1 month after primaryimmunization. The titers increased to >1 million, 1 month after thefirst dose of booster, even in the case of the single primary doseunresponsive animal [FIG. 4 b], reaching an antibody titer of more than2 million following the second booster injection. The IgG subtypes werealso tested with various bleeds. Three out of five animals showedpredominantly IgG1 compared to IgG2, and for the other two animals, thereverse was the case [FIG. 4 c]. IgG3 was totally absent from immunizedsera in all the bleeds tested.

Mosquito MFA showed strong transmission blocking activity by all animalsfor various bleeds obtained at different time points post immunization[Table 2, shown below].

TABLE 2 Bleeds [% transmission blocking * (infected/total mosquito)]Animals Dec. 10, 2007 Jan. 10, 2008 Feb. 21, 2008 Mar. 06, 2008 May 05,2008 Pan 3104 92.8 (14/27) 95.7 (11/25) 98.7 (6/22) 97.7 (7/27) 98.8(4/19) Pan 3140 94.4 (14/21) 97.3 (12/28) 98.1 (6/24) 98.4 (7/23) 97.8(7/18) Pan 3163 98.1 (5/19)  98.5 (5/18)  97.3 (9/27) 97.3 (7/20)  97.4(11/24) Pan 3275 88.1 (12/20) 95.3 (11/25)  96.2 (10/28) 96.2 (9/23)97.6 (5/22) Pan 3313 91.8 (13/25) 97.0 (9/24)   94.7 (13/27) 97.8 (8/20) 95.3 (12/24) *P < 0.0001 (Mann-Whitney test)

Table 2 shows transmission blocking activity of sera of different bleedsat various time points from baboons immunized with CH-rPfs48/45 inMontanide ISA-51. MFA was done with sera collected on Dec. 10, 2007 (1mo post primary immunization), Jan. 10, 2008 (1 mo post 1st boost, Feb.21, 2008 (15 d post 2nd boost), Mar. 6, 2008 (1 mo post 2nd boost), andMay 5, 2008 (3 mo post 2nd boost). The geometric mean of oocystnumbers/midgut in the presence of individual pre-immune sera were 22.24(Pan 3104), 18.43 (Pan 3140), 22.1 (Pan 3163), 6.31 (Pan 3275), and 19.7(Pan 3313), respectively. Numbers within parenthesis represent totalnumber of infected mosquitoes/total number of mosquitoes dissected foreach feed.

Even after primary immunization, the sera (at 1:2 dilution) were capableof impressive transmission blocking with more than 93% average reductionin the number of oocysts in comparison to pre-immune sera ofcorresponding animal. Pre-immune sera from each animal were used as ameasure of 100% transmission for the corresponding test sera. Pre-immuneserum from one animal (Pan 3275) exhibited ˜30% reduced transmissionwhen compared with the pre-immune sera of other four baboons. It ispossible that natural infection by other Plasmodium-like parasites, suchas Enteropoides and Hepatocystis spp might elicit partly cross-reactiveand inhibitory activity. The average transmission blocking activityincreased to greater than 97% in all the animals after a booster dose.In order to titrate the blocking effectiveness of immune sera from theseanimals, sera obtained one month after the second boost were furthertested at various dilutions (1:4, 1:8, 1:16) in MFA. While exhibitingstrong blocking activity at 1:4 and 1:8 dilutions, the sera at 1:16dilution were still able to reduce transmission (ranging from 74% to86%).

After the second booster dose, all five animals were bled at monthlyintervals and sera analyzed for antibody titers by ELISA and functionaltransmission blocking activity in MFA. As shown in FIG. 5, high antibodytiters and high transmission blocking activities were maintained formore than 7 months suggesting further long lasting nature of immuneresponses elicited by CH-rPfs48/45 in nonhuman primates.

The data reported herein shows for the very first time the expression offull length soluble recombinant Pfs48/45 protein in proper conformationwith high yield following application of the codon harmonizationapproach for recoding gene sequences. The recombinant protein(designated CH-rPfs48/45) showed remarkable immunogenicity andfunctional activity, i.e. transmission blocking activity in miceimmunized in the three adjuvant formulations tested. Furthersignificance of the results is reflected by evidence from pre-clinicalvaccine testing in nonhuman primates. The vaccine revealed potentimmunogenicity and effective blocking activity even after a singleimmunization which became much stronger (approaching 100% reduction)after a booster immunization. Ease of expression and purification ofprotein at high yields, conformational folding and strong immunogenicityof CH-rPfs48/45 in mice and non human primates provide a much neededrationale for moving ahead with the development of malaria TBV based onPfs48/45 antigen. Mosquito infection (oocyst load and percent infectedmosquitoes) in the presence of 4/5 pre-immune sera were comparable tothose obtained with normal human serum negative control.

Expression of P. falciparum proteins in heterologous hosts is especiallychallenging due to high A/T content within protein coding genes (>80%).All previous attempts to express properly folded full length recombinantPfs48/45 have remained unsuccessful. As reported herein, codonharmonization was used to express Pfs48/45 in E. coli in a functionallycorrect conformation. Another important consideration for thedevelopment of a transmission blocking vaccine is the production ofproteins in correctly folded conformation. The results herein report arobust expression of Pfs48/45 after codon harmonization of the codingsequence, the purified protein was found to be in correct conformationas revealed by Western blot analysis using monoclonal antibodiesdirected against reduction-sensitive conformational epitopes. Therecombinant Pfs48/45 was recognized by the same antibodies even aftertreatment with reducing agents, suggesting that the target epitopes inthe expressed proteins are stable and not susceptible to reduction.

Further development of a transmission blocking vaccine based on Pfs48/45is supported by the observation that the purified protein exhibited verystrong and longer lasting functional immunogenicity in baboons. Theantibodies elicited by vaccination continued to effectively suppress,even 5-6 months after the last booster immunization, infectivity (bothoocyst burden and percentage of mosquitoes infected) of P. falciparumgametocytes in Anopheles mosquitoes. Previous studies have shown thatPfs48/45 proteins normally expressed in the erythrocytic gametocytestage of the parasite is also a target of partially effective naturalimmune response. It has been hypothesized that a vaccine inducedresponse could be further boosted during natural infection and thus helpin maintaining higher antibody levels in the vaccinated people.

Methods

The invention was performed with, but not limited to, the followingmethods and materials.

Cloning, Expression and Purification of CH-rPfs48/45

The codon harmonized sequence of Pfs48/45 containing 6× Histidines atN-terminal end was synthesized by Retrogen Inc. and cloned into theexpression vector pET(K-) in E. coli BL21 (DE3) competent cells(Invitrogen Corp.). The cells containing pET(K-)Pfs48/45 were grownovernight at 30° C. in LB media containing 1% glucose and 50 μg/mlKanamycin. The overnight culture was diluted 100 fold into 1 l culturein above mentioned media and grown at 30° C. with agitation until theOD600 of the culture reached 1.00. The cells were then induced with 0.1mM of IPTG and grown for 3 h at 30° C. The cells were then harvested andcentrifuged at 3800×g at 4° C. for 20 min. The pellet was kept at −80°C. for further processing. The frozen pellet was resuspended in 1×PBS(pH 7.4) at a pellet to buffer ration of 1:10 (w/v) and was lysed bymicrofluidization (Model M110Y, Microfluidics). The lysate wascentrifuged at 24000×g for 45 min at 4° C. The lysate pellet wasresuspended in 1×PBS containing 1% Tween-80 (final v/v), extracted for30 min at 22° C. and centrifuged at 24000×g for 30 min at 4° C. Thepellet was resuspended in 1×PBS containing 0.5% Sodium Lauroyl Sarcosine(sarcosyl) (final v/v), extracted and centrifuged as before. Thesupernatant was then passed through Ni-NTA agarose column (QIAGEN)according to manufacturer's protocol. The protein was eluted as 1 mlfractions with 1 M imidazole in 1×PBS as elution buffer. The protein wasdialyzed against 1×PBS (pH 7.4) containing 10% glycerol and 0.2%Tween-80. Finally, the protein content was estimated using BCA™ ProteinAssay kit (Pierce) and the endotoxin level in the protein was measuredusing QCL-1000 Endpoint chromogenic LAL assay kit (Lonza).

Characterization of CH-rPfs48/45

The CH-rPfs48/45 was characterized by Western blot analysis. Briefly,samples from each purification step described above were run on SDS-PAGEand transferred to nitrocellulose membrane (Bio-Rad). The membrane wasblocked overnight with 1×PBS containing 5% non-fat dry milk and 0.1%Tween-20 (blocking buffer) at 4° C. Following blocking, the membrane waswashed with 1×PBS containing 0.1% Tween-20 (wash buffer) and incubatedwith either 6×His mAb (Clontech) at 1:1000 dilution [FIGS. 1 d, e] orIIC5B10 mAb (MR4) at 1:5000 dilution [FIG. 1 f] for 1 h at 22° C. Themembrane was washed 4× in wash buffer for 30 min at 22° C. and incubatedwith HRP-conjugated anti-mouse IgG mAb (GE Healthcare) at 1:10000dilution in blocking buffer for 1 h at 22° C. This was followed bywashing 4× with wash buffer and ECL Plus chemiluminescent substrate (GEHealthcare) was used as detection reagent.

Immunization of Mice

Groups (n=5) of female BALB/c mice were immunized with 10 μg ofCH-rPfs48/45 emulsified in either Complete Freund's Adjuvant (CFA)(Sigma) or Montanide ISA-51 (SEPPIC) or mixed in aluminum hydroxide(Alhydrogel, Brenntag) adjuvant through the intraperitoneal route. Themice immunized in CFA were boosted at 4 week intervals twice with thesame quantity of protein in incomplete Freund's adjuvant. Mice in theother adjuvant groups were boosted at 4 week intervals thrice inMontanide ISA-51 or Alum, respectively. Groups of control mice wereimmunized with adjuvant formulations only. Blood was collected on day 0(Pre-immune sera) and 4 weeks after primary immunization and 2 weekspost each boost for analysis of anti-Pfs48/45 IgG titer.

Immunization of Olive Baboons (Papio anubis)

The vaccine trial in baboons was approved by the institutional andscientific review committee of the Institute of Primate Research with aprotocol #Oct. 10, 2007. Because these animals were trapped from theirwild habitats, they were quarantined for 3 months and screened for thepresence of any worms and protozoan parasites and successfully treatedappropriately, if found infected, prior to initiating vaccination.Moreover, animals were also screened by three intradermal tuberculintests and found to be negative for mycobacterial infections. Detailedhematological tests were also administered on all five animals duringtheir quarantine period, just prior to and at the termination of thevaccine trial, and were certified to be in excellent health at all timepoints with no observable trial-related effects. A group of five baboons(ranging 7.6 to 12.2 kg in body weight) were immunized with 50 μg ofCH-rPfs48/45 in Montanide ISA-51, water-in-oil emulsion. The animalswere sedated with ketamine (10 mg/kg) for immunization and bloodcollection as per the schedule described in FIG. 4 a.

ELISA

To assess the immunogenicity of CH-rPfs48/45, ELISA was done. Briefly,Immulon-2 plates were coated with 1.5 μg/ml CH-rPfs48/45 incarbonate-bicarbonate buffer (pH 9.6) overnight at 4° C., blocked with5% milk in PBS and incubated with various dilutions of sera at 37° C.for 1 h. The plates were washed 5 times in PBS-0.05% Tween-20 (PBST)followed by further incubation with 1:10000 dilution of horseradishperoxidase conjugated anti-mouse IgG antibody for 1 hour at 37° C. Afterwashing in PBST, wells were developed using ABTS substrate (Kirkegaard &Perry Laboratories Inc.) for 20 min at 22° C. and read at 405 nm in theELISA reader. Anti-Pfs48/45 whole IgG end point titers were calculatedfrom the highest group mean reciprocal serum dilution greater than themean plus 3 standard deviations OD reading of pooled pre-immune sera ineach assay. For IgG subtype analysis, mouse sera were tested at a single1:1000 dilution. Various isotype-specific secondary antibodies used wereanti-mouse IgG1, IgG2a, IgG2b and IgG3 from Kirkegaard & PerryLaboratories Inc.

The ELISA with baboon sera was done following a similar protocol andendpoint titers were calculated using the same criteria. The secondaryantibody was anti-human IgG1, IgG2, and IgG3 conjugated to peroxidase(The Binding Site, Birmingham, UK) and used at 1:5000 dilution. Varioussera were tested at 1:5000 dilution for IgG subclass analysis.

Parasite

Plasmodium falciparum NF54 parasites were maintained using normal redblood cells and normal human serum (O+ve blood group) as s described[32]. Stage V gametocytes were used in live IFA studies and membranefeeding assays. To extract gametocytes for Western blot analysis, thegametocyte culture was centrifuged at 1000×g for 5 min at 22° C. The RBCwas lysed with 0.15% saponin in PBS for 5 min at 22° C. The gametocyteswere collected after centrifugation at 1800×g for 5 min and washedthrice in 1×PBS. The gametocytes were resuspended in 25 mM Tris-Cl (pH7.5) containing 150 mM NaCl and 1× protease inhibitor cocktail and keptfrozen at −70° C. till use.

Live IFA

The gametocytes were harvested from 19 day culture and gametes wereproduced by incubating the gametocytes in exflagellation buffer andpurified by discontinuous Nycodenz gradient centrifugation as describedpreviously [33]. Extracellular gametes were incubated at 4° C. with1:100 to 1:1000 dilution of immune murine sera for 60 min. Parasiteswere washed 3 times with 1% BSA in PBS followed by incubation withFITC-anti mouse antisera (Alexa fluor 488), 1:500 dilution at 4° C. for60 minutes. After washing, cells were examined by upright fluorescentNikon E800 microscope (Japan) at 100× magnification.

Membrane Feeding Assay

To test the transmission blocking activity, the murine and baboon immunesera were mixed with cultured P. falciparum (NF54) stage V gametocytes,normal red blood cells and normal human sera (donor blood group: O+) andfed to Anopheles gambiae (starved for 5-6 hours) mosquitoes throughwater jacketed glass membrane (stretched parafilm) feeders [10,33]Briefly, washed human red blood cells and cultures containing stage Vgametocytes were resuspended to 66% hematocrit and 0.3% gametocytemia innormal human serum and maintained throughout at 37° C. Fifty microlitersof various test sera (appropriately diluted in normal human sera) weremixed with 150 μl of resuspended gametocyte mix and quickly added toindividual membrane feeders placed on top of cups containing starvedmosquitoes. The mosquitoes were allowed to engorge for 15 min. The unfedmosquitoes were separated within 1 h of feeding and blood fed mosquitoes(typically 25-30 per cup) were maintained in the insectary at 26° C. at80% relative humidity. In certain cases the total number of blood fedmosquites was less than 25 due to poor feeding. Moreover, a small numberof blood fed mosquites (less than 5%) did not survive 9-10 daysincubation period prior to dissection. Midguts were dissected 9-10 daysafter blood meal for enumeration of oocysts after staining with 1%mercurochrome. Transmission blocking activity of individual sera wascalculated as percentage of reduction in oocyst number per midgut withtest sera in comparison with pooled pre-immune sera (pre-immune murineor baboon sera was taken as allowing 100% transmission).

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

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1. A method of blocking transmission of Plasmodium falciparum orPlasmodium vivax in a subject comprising: administering to a subject animmunogenic composition comprising one or more Plasmodium falciparum orPlasmodium vivax pre-fertilization antigens, thereby blockingtransmission of Plasmodium falciparum or Plasmodium vivax in thesubject.
 2. A method of immunizing a subject against Plasmodiumfalciparum or Plasmodium vivax comprising: administering to a subject animmunogenic composition comprising one or more Plasmodium falciparum orPlasmodium vivax pre-fertilization antigens, thereby immunizing thesubject against Plasmodium falciparum or Plasmodium vivax.
 3. A methodfor treating or preventing malaria in a subject comprising:administering to a subject an immunogenic composition comprising one ormore Plasmodium falciparum or Plasmodium vivax pre-fertilizationantigens, thereby preventing malaria in the subject.
 4. A method ofblocking transmission of Plasmodium falciparum or Plasmodium vivax in asubject comprising: administering to a subject an immunogeniccomposition comprising one or more Plasmodium falciparum or Plasmodiumvivax post-fertilization antigens, thereby blocking transmission ofPlasmodium falciparum or Plasmodium vivax in the subject.
 5. A method ofimmunizing a subject against Plasmodium falciparum or Plasmodium vivaxcomprising: administering to a subject an immunogenic compositioncomprising one or more Plasmodium falciparum or Plasmodium vivaxpost-fertilization antigens, thereby immunizing the subject againstPlasmodium falciparum or Plasmodium vivax.
 6. A method for treating orpreventing malaria in a subject comprising: administering to a subjectan immunogenic composition comprising one or more Plasmodium falciparumor Plasmodium vivax post-fertilization antigens, thereby preventingmalaria in the subject.
 7. The method of claim 1, wherein the one ormore pre-fertilization or post-fertilization antigens are selected fromthe group consisting of Pfs48/45, Pfs 230, Pfs25, Pvs48/45, Pvs 230 andPvs25. 8-9. (canceled)
 10. The method of claim 1, wherein the one ormore pre-fertilization antigens or post-fertilization antigens isderived from a codon harmonized gene.
 11. The method of claim 10,wherein the codon harmonized gene is encoded by the amino acid sequencecorresponding to SEQ ID NO: 1, or the codon harmonized gene is encodedby the amino acid sequence corresponding to SEQ ID NO: 3, or the codonharmonized gene is encoded by the amino acid sequence corresponding toSEQ ID NO:
 5. 12-13. (canceled)
 14. A method of blocking transmission ofPlasmodium falciparum or Plasmodium vivax in a subject comprising:administering to a subject an immunogenic composition comprising one ormore Plasmodium falciparum or Plasmodium vivax surface antigens, therebyblocking transmission of Plasmodium falciparum or Plasmodium vivax inthe subject, or A method of immunizing a subject against Plasmodiumfalciparum or Plasmodium vivax comprising: administering to a subject animmunogenic composition comprising one or more Plasmodium falciparum orPlasmodium vivax surface antigens, thereby immunizing the subjectagainst Plasmodium falciparum or Plasmodium vivax, or A method fortreating or preventing malaria in a subject comprising: administering toa subject an immunogenic composition comprising one or more Plasmodiumfalciparum or Plasmodium vivax surface antigens, thereby preventingmalaria in the subject. 15-16. (canceled)
 17. The method of claim 14,wherein the surface antigens are gametocyte or gamete surface antigens.18. The method of claim 17, wherein the gametocyte or gamete surfaceantigens are selected from the group consisting of: Pfs48/45, Pfs230,Pvs48/45 and Pvs230. 19-35. (canceled)
 36. An immunogenic compositioncomprising one or more pre-fertilization or post-fertilization antigensfrom P. falciparium or P. vivax.
 37. The immunogenic composition ofclaim 36, wherein the one or more pre-fertilization orpost-fertilization antigens are selected from the group consisting ofPfs48/45, Pfs230, Pfs 28, Pfs25, Pvs48/45, Pvs 230, Pvs 28, and Pvs25.38-44. (canceled)
 45. A vector comprising a codon harmonized Pfs48/45sequence suitable for expression in a cell, or A vector comprising acodon harmonized Pvs48/45 sequence suitable for expression in a cell, orA vector comprising a codon harmonized Pfs25 sequence suitable forexpression in a cell. 46-50. (canceled)
 51. A cell expressing the vectorof claim
 46. 52. (canceled)
 53. A kit comprising an immunogeniccomposition comprising one or more Plasmodium falciparum or Plasmodiumvivax pre-fertilization or post-fertilization antigens, and instructionsfor use in reducing transmission of Plasmodium falciparum or Plasmodiumvivax, or A kit comprising an immunogenic composition comprising one ormore Plasmodium falciparum or Plasmodium vivax pre-fertilization orpost-fertilization antigens, and instructions for use in preventingmalaria. 54-56. (canceled)
 57. A method for preparing a codon harmonizedpre-fertilization or post-fertilization antigen sequence encoded by a P.falciparum or P. vivax pre-fertilization or post-fertilization genecomprising: determining the frequency of codon usage of thepre-fertilization or post-fertilization gene coding sequence; andsubstituting codons in the coding sequence with codons of similarfrequency from a host cell which code for the same pre-fertilization orpost-fertilization antigen, thereby preparing a codon harmonizedpre-fertilization or post-fertilization antigen sequence, or A methodfor preparing a codon harmonized Pfs48/45 antigen sequence encoded by apre-fertilization or post-fertilization gene comprising: determining thefrequency of codon usage of the pre-fertilization or post-fertilizationgene coding sequence, wherein the Pfs48/45 sequence corresponding to thenucleic acid sequence represented by SEQ ID NO: 7; and substitutingcodons in the coding sequence of SEQ ID NO: 7 with codons of similarfrequency from a host cell which code for the Pfs48/45 antigen, therebypreparing a codon harmonized Pfs48/45 antigen sequence. 58-61.(canceled)
 62. A codon harmonized nucleotide sequence prepared accordingto the method of claim 57.